Product of heat treatment of uronic acid, food, drink, or drug including the product

ABSTRACT

A product obtained by heating at least one substance selected from the following (a), (b) and (c). 
     (a) uronic acid or uronic acid derivative; 
     (b) a saccharide compound containing uronic acid or a saccharide compound containing uronic acid derivative; and 
     (c) a substance containing a saccharide compound containing uronic acid or a substance containing a saccharide compound containing uronic acid derivative; 
     and food, beverage or a pharmaceutical agent which is characterized in containing the above-mentioned heat-treated product.

BACKGROUND OF THE INVENTION

1. Field of the Invention

An object of the present invention is to develop a product containing ahighly safe and physiologically active substance having an anticanceraction, an apoptosis-inducing action, and the like, and to offer afunctional food or beverage exhibiting a high physiological effectcontaining said product. The present invention also offers antibacterialagents, dentifrices, antiseptic agents, apoptosis inducers, anticanceragents and antiulcer agents containing said product as an effectivecomponent. The present invention further offers a method for inducingapoptosis where said method is useful, for example, in elucidating themechanism of apoptosis and in screening the apoptosis inhibitors. Thepresent invention still further offers a method for the manufacture of aproduct containing the physiologically active substance of the presentinvention.

2. Description of the Related Art

In recent years, a phenomenon called “apoptosis” which is aself-destructive cell death or a suicidal cell death has attractedattentions regarding the death of cell tissues.

Unlike a necrosis which is a pathological cell death, an apoptosis isconsidered to be the death which is inherently programmed in genes ofthe cells themselves. Thus, it is believed that some external orinternal factors trigger the activation of genes which program theapoptosis whereby a programmed death gene protein is biosynthesizedbased upon the genes and the cells themselves are decomposed by theresulting programmed death gene protein whereby the death is resulted.

If such an apoptosis can be expressed in a desired tissue or cell, itwill be quite meaningful because unnecessary or pathogenic cells such ascancer cells can be eliminated from the living body in a natural manner.

SUMMARY OF THE INVENTION

An object of the present invention is to develop a product containing ahighly safe and physiologically active substance having an anticanceraction, an apoptosis-inducing action, and the like whereby a method forthe manufacture of said product and also food or beverage containingsaid product are offered. Another object of the present invention is tooffer pharmaceuticals such as antibacterial agents and apoptosisinducers containing said compound and to offer a method of inducing anapoptosis using said product as an effective component.

An outline of the present invention will be as follows. Thus, the firstaspect of the present invention is a product obtained by heating atleast one substance selected from the following (a), (b) and (c).

(a) uronic acid or uronic acid derivative;

(b) a saccharide compound containing uronic acid or a saccharidecompound containing uronic acid derivative; and

(c) a substance containing a saccharide compound containing uronic acidor a substance containing a saccharide compound containing uronic acidderivative.

The second aspect of the present invention is a method for themanufacture of a heat-treated product, characterized in that, saidmethod includes a step of heating at least one substance selected fromthe following (a), (b) and (c).

(a) uronic acid or uronic acid derivative;

(b) a saccharide compound containing uronic acid or a saccharidecompound containing uronic acid derivative; and

(c) a substance containing a saccharide compound containing uronic acidor a substance containing a saccharide compound containing uronic acidderivative.

The present inventors have found that a heat-treated product(hereinafter, said product will be referred to as a “heat-treatedproduct of the present invention”) of at least one substance selectedfrom uronic acid, uronic acid derivative, a saccharide compoundcontaining uronic acid, a saccharide compound containing uronic acidderivative, a substance containing a saccharide compound containinguronic acid and a substance containing a saccharide compound containinguronic acid derivative has a potent anticancer action,apoptosis-inducing action, antibacterial action and antiulcer actionwhereby the present invention has been achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an action of a heat-treated product of pectin to cancercells;

FIG. 2 shows an action to cancer cells of the samples before and afterdialysis;

FIG. 3 shows an action to cancer cells of the filtrate obtained byultrafiltration;

FIG. 4 shows an action to cancer cells of the fraction obtained by a gelfiltration;

FIG. 5 shows an action to cancer cells of the heat-treated products ofuronic acids;

FIG. 6 shows a relation between the pH when uronic acid is heated andthe action of the heat-treated product to cancer cells;

FIG. 7 shows an action to cancer cells of a product obtained by heatingpectin under an acidic condition;

FIG. 8 shows an action to cancer cells of a fraction obtained by solventextraction of a product obtained by heating pectin under an acidiccondition;

FIG. 9 shows an action to cancer cells of a product obtained by heatingpectin firstly under an alkaline condition and then under an acidiccondition;

FIG. 10 shows an action to cancer cells of a product obtained by heatinggalacturonic acid under an acidic condition;

FIG. 11 shows an action to cancer cells of a product obtained by heatingglucuronic acid under an acidic condition;

FIG. 12 shows an action of a heat-treated solution I of pectin to cancercells;

FIG. 13 shows a relation between the dilution rate of a heat-treatedproduct of glucuronic acid and the survival rate of the cells;

FIG. 14 shows an action of a heat-treated product of alginic acids tocancer cells;

FIG. 15 shows an anticancer action of a heat-treated product of pectinto a leukemia cell line;

FIG. 16 shows an anticancer action of a heat-treated product of uronicacids to a leukemia cell line; and

FIG. 17 shows a differentiation-inducing action of a heat-treatedproduct of uronic acid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be illustrated in a specific manner ashereinafter.

In the present invention, there is no particular limitation for uronicacid, uronic acid derivative, a saccharide compound containing uronicacid, a saccharide compound containing uronic acid derivative, asubstance containing a saccharide compound containing uronic acid and asubstance containing a saccharide compound containing uronic acidderivative provided that the product obtained by heating them exhibitsanticancer action, apoptosis-inducing actions, and the like, and thatanticancer substance and/or apoptosis-inducing substance are/is producedin said heat-treated product.

Uronic acid is sometimes called glycuronic acid and is a general namefor hydroxyaldehyde carboxylic acids in which an aldehyde group onaldose remains as it is while only a primary alcohol group at anotherend is oxidized to a carboxyl group. It is present in nature as aconstituting component for various polysaccharides of animals andplants. Examples of the polysaccharides containing uronic acids arepectin, pectic acid, alginic acid, hyaluronic acid, heparin, fucoidan,chondroitin sulfate, dermatan sulfate, and the like, and they have beenknown to exhibit various physiological functions.

There is no particular limitation for the uronic acid used in thepresent invention. Thus, examples of the uronic acid are galacturonicacid, glucuronic acid, guluronic acid, mannuronic acid and iduronic acidwhile examples of the uronic acid derivative are lactones, esters,amides, salts, and the like, of the above-mentioned ones and anysubstance which produces anticancer substance and/or apoptosis-inducingsubstance by heat treatment is covered by the derivative of the presentinvention. Examples of the uronic acid lactone are glucurono-6,3-lactone(hereinafter, abbreviated as glucuronolactone), mannurono-6,3-lactoneand idurono-6,3-lactone. Examples of the uronic acid ester are methyl,ethyl, propylene glycol and carboxymethyl uronates which can bemanufactured from uronic acid. Uronic acid amide can be manufactured byamidation of uronic acid. Salts of them can be manufactured by commonmethods.

The saccharide compound containing uronic acid or uronic acid derivativeof the present invention means a saccharide compound containing uronicacid and/or uronic acid derivative and there is no particular limitationtherefor. Thus it covers, for example, pectin, pectic acid, alginicacid, hyaluronic acid, heparin, fucoidan, chondroitin sulfate,chondroitin and dermatan sulfate including decomposed products,derivatives of the decomposed products and salts of the decomposedproducts thereof which are chemically, enzymatically orphysically-treated products thereof.

In the above-mentioned chemical treatment, the starting saccharidecompound is, for example, treated at room temperature to 200° C. forseveral seconds to several hours or, preferably, at 50-130° C. forseveral seconds to an hour (in the case of pectin, treated for exampleat pH 6.8, 95° C. for several minutes to several tens minutes) whereupona beta-elimination takes place to give a saccharide compound havingunsaturated uronic acid and/or unsaturated uronic acid ester in which anabsorbance at around 235 nm is increased. The saccharide compound of thepresent invention covers a saccharide compound containing unsaturateduronic acid and/or unsaturated uronic acid ester at a non-reducing endprepared by a beta-elimination of a polysaccharide compound containinguronic acid and/or uronic acid ester.

An example of the above-mentioned enzymatic treatment is a knowndecomposition method in which the starting saccharide compoundcontaining uronic acid and/or uronic acid ester is decomposed by ahydrolase for the saccharide containing uronic acid and/or uronic acidester. Another example is a known decomposition method in which thesaccharide containing uronic acid and/or uronic acid ester is decomposedby a lyase for the saccharide containing uronic acid and/or uronic acidester. For example, in the case of pectin or pectic acid, adecomposition is conducted by a known pectin lyase (EC 4.2.2.10), pecticacid lyase (EC 4.2.2.2) or exopolygalacturonic acid lyase (EC 4.2.2.9)to give a saccharide compound having 4-deoxy-L-threo-hex-4-enopyranosyluronate or methyl ester thereof at a non-reducing end. In the case ofhyaluronic acid, a hyaluronate lyase (EC 4.2.2.1) is used while, in thecase of alginic acid, an alginate lyase (EC 4.2.2.3) is used. Theenzymatically decomposed products having4-deoxy-L-threo-hex-4-enopyranosyl uronate or methyl ester thereof atthe non-reducing end prepared as such are covered by the saccharidecompound of the present invention as well.

Examples of the above-mentioned physical treatment are the treatment ofthe starting saccharide compound with near infrared ray, infrared ray,microwave, ultrasonic wave, and the like. Thus, for example, pectinand/or pectic acid are/is placed in a neutral (in terms of pH) or analkaline solution and subjected to an ultrasonic wave for applying avibrational energy at an appropriate temperature of not lower than roomtemperature under an appropriate reductive operation in the presence of,for example, ascorbic acid for not shorter than one second or,preferably, from five seconds to one hour. Besides the ultrasonic wave,it is also effective to irradiate with microwave, near infrared ray,infrared ray, and the like, or a combination thereof The irradiation maybe conducted either continuously or intermittently.

In addition, in the present invention, a substance which contains theabove-mentioned saccharide compound containing uronic acid and/or itsderivative such as fruit, rind of a fruit, strained lees of a fruit,vegetable, strained lees of a vegetable, sea algae, and the like, may beused either as it is or after being dried and crushed. Further, a liquidof the saccharide compound containing uronic acid and/or its derivativeobtained by extracting the above-mentioned substance which contains asaccharide compound containing uronic acid and/or its derivative, or apurified substance obtained from said extracted liquid may be used aswell. Preparation of such an extracted liquid of the saccharide compoundcontaining uronic acid and/or its derivative and purification from theextracted liquid may be conducted by known methods and there is noparticular limitation therefor.

Examples of the substance which contains the saccharide compoundcontaining uronic acid or uronic acid ester are as follows. Thus,fruits, vegetables, leaves, seeds, and the like of dicotyledons such asapple, citrus fruits (e.g., mandarin orange and lemon), banana, nappacabbage, cabbage, lettuce, perilla, pumpkin, celery, burdock, echalote,broccoli, green pepper, spinach, carrot, leaves of carrot, leaves ofdaikon (Japanese radish), tea leaves, sesame, beans, potato, and thelike; cereals of monocotyledons such as wheat and rice; algae such asbrown algae (e.g., sea tangle and wakame seaweed), red algae, greenalgae and unicellular green algae; microorganisms such as Basidiomycetes(e.g., Lyophyllum ulmarium, Lyophyllum decastes, Pholiota nameko,Cortinellus shiitake, Flammulina verutipes, Agaricus ostreatus andPasalliota campestris), Ascomycetes (e.g., Cordyceps militaris and otherCordyceps sp.), yeasts, filamentous fungi (e.g., Aspergillus sp.) andbacteria (e.g., Bacillus natto and lactic acid bacteria); and animalssuch as vertebrates and invertebrates. In the present invention, asubstance which contains a saccharide compound containing uronic acidand/or uronic acid derivatives derived from the above-mentioned plants,microorganisms or animals may be used.

The polysaccharides which are saccharide compounds containing uronicacid and/or uronic acid derivatives can be manufactured by knownchemical, enzymatic or physical methods. In the case of pectin forexample, a high-molecular polysaccharide extracted from, for example,rind of citrus fruits or apple may be used. Materials for themanufacture of pectin on an industrial scale are fruits and, in additionto strained lees (mostly comprising endocarp) after preparing juice ofcitrus fruits such as lemon and lime, the strained lees afterpreparation of apple juice is used as well. Such strained lees mostlycontain insoluble protopectin and it is solubilized (extracted) duringthe course of manufacture to prepare pectin. Solubilization can beconducted by extracting with acidic warm to hot water and, when theconditions such as temperature, pH and time are properly controlleddepending upon the type of the starting material, it is possible tomanufacture pectin having predetermined molecular weight and degree ofesterification in a high yield. The extract is purified by means ofcentrifugation or filtration and concentrated and alcohol is addedthereto whereupon pectin can be precipitated and recovered. Therecovered precipitate is dried and crushed to prepare a dry pectin.

The main structure of pectin is a partially methylated galacturonic acidpolymer. The carboxyl group is either methylated, left as a free acid ormade into a salt such as ammonium salt, potassium salt or sodium salt.Depending upon the degree of methylation (DM; ratio of methoxyl groupsto total carboxyl groups), pectin is classified into an HM pectin havinga high DM and an LM pectin having a low DM [“Handbook of Materials forDeveloping New Food Products” edited by Satoshi Yoshizumi, et al.,published by K. K. Korin, pages 114-119 (1991)] and, in the presentinvention, pectin which is commercially available as a food additive[“Handbook of Natural Products” edited by Akio Toyama, et al., publishedby Shokuhin To Kagakusha, 12th Edition, page 138 (1993)], commerciallyavailable HM pectin and LM pectin, and the like, [refer to theabove-mentioned “Handbook of Materials for Developing New FoodProducts”] may be used.

Decomposed product of a saccharide compound containing uronic acidand/or uronic acid derivative may be manufactured by known chemical,enzymatic or physical treating methods. Uronic acid, uronic acidderivatives, oligosaccharides, and the like which are manufactured bysynthetic means are also covered by the present invention.

The heat-treated product which is used in the present invention may bemanufactured from a material selected from (a) uronic acid or uronicacid derivative; (b) a saccharide compound containing uronic acid or asaccharide compound containing uronic acid derivative; and (c) asubstance containing a saccharide compound containing uronic acid or asubstance containing a saccharide compound containing uronic acidderivative.

With regard to a method for the heating treatment in the manufacture ofthe heat-treated product of the present invention, uronic acid, uronicacid derivative, a saccharide compound containing uronic acid,saccharide compound containing uronic acid derivative, a substancecontaining a saccharide compound containing uronic acid and/or asubstance containing a saccharide compound containing uronic acidderivative are/is heated, for example, at 60-350° C. for several secondsto several days or, preferably, at 80-150° C. for several minutes toseveral days. In the case of pectin, a heat-treated product having aphysiological activity such as anticancer action or apoptosis-inducingaction can be prepared by heating the pectin, for example, at 80-150° C.for several minutes to several days while, in the case of uronic acids,uronic acid lactones and uronic acid esters, desired heat-treatedproduct can be prepared by heating them at 60-150° C. for severalminutes to several days.

Although there is no particular limitation for the pH during the heatingtreatment, it is preferred to conduct the heating under neutral toacidic conditions and, depending upon the material used, the pH duringthe heating may be adjusted. Usually, however, production ofphysiologically active substances such as anticancer substance,apoptosis-inducing substance, and the like is promoted by heating underan acidic condition.

There is no particular limitation for the concentration of the materialupon heating provided that the concentration is within such a range thatthe physiologically active substances such as anticancer substance,apoptosis-inducing substance, and the like, can be produced by theheating treatment. Thus, the concentration may be decided by takingworkability, yield, and the like into consideration.

The heating treatment in the present invention may be either wet heatingor dry heating. In the case of a wet heating, any of wet heating methodssuch as heating with steam, heating with steam under high pressure,heating under high pressure, and the like, may be used while, in thecase of a dry heating, any of dry heating methods such as a directheating using dry and hot air and an indirect heating from a heat sourcethrough a partition may be used. Examples of the direct heating are adry heating by an air stream and a dry heating by means of sprayingwhile those of the indirect heating are a dry heating by means of adrum, and the like. In addition, the material for the heating treatmentof the present invention may be treated by any of common heating methodssuch as boiling, toasting, roasting, decocting, steaming, frizzling,flying, and the like.

The heat-treated product of the present invention is a heat-treatedproduct obtained by the above-mentioned heating methods and a fractioncontaining a physiologically active substance in said heat-treatedproduct.

The heat-treated product of the present invention contains two or moresubstances which showapoptosis-inducing action, anticancer action,antibacterial action, antiviral action, and the like. In addition,reductons having antoxidative action are also produced during theheating treatment of the present invention. Therefore, when theconditions for the heating treatment are changed according to theobject, it is possible to prepare the heat-treated product of thepresent invention having a desired substandce, The heat-treated produtof the present invention can be fractionated using its physiologicalactivity as an index. For example, the molecular weight fractionation ofthe heat-treated product is conducted by a known method such as gelfiltration or fractionation using a molecular weight fractionatingmembrane to prepare each molecular weight fraction whereupon thegeat-treated product of the present invention having a high activity canbe prepared. Further, a desired fraction can be also prepared by solventextraction, fractional distillation and various chromatographic methodsusing ion exchange resin, and the like.

Examples of gel filtration are that, when Cellulofine GCL-300 is used,it is possible to prepare any of the molecular weight fractions such asthose where the molecular weight (MW) is MW>25,000; 25,000≧MW>10,000;10,000≧MW>5,000; and 5,000≧MW while, when Cellulofine GCL-25 is used, itis possible, for example, to fractionate a fraction of 5,000≧MW into anyof the molecular weight fractions such as 5,000 2≧MW >3,000;3,000≧MW>2,000; 2,000≧MW>1,000; 1,000≧MW>500; and 500≧MW.

When an ultrafiltration membrane is used, the molecular weightfractionation can be conducted on an industrialscale. For example, whenFE10-FUSO382 (manufactured by Daicel) is used, it is possible to preparea fraction having a molecular weight of 30,000 and less while, whenFE-FUS-T653 (manufactured by the same company) is used, it is possibleto prepare a fraction having a molecular weight of 6,000 and less.Further, the use of a nanofilter membrane is able to give a fractionhaving a molecular weight of 500 or less. When the above-mentioned gelfiltration and molecular weight fractionation are combined, any of themolecular weight fractions may be prepared.

In the heat-treated product of the present invention, the fractionhaving a molecular weight of 30,000 or less has strong anticancer andapoptosis-inducing activities and, particularly, the fraction having amolecular weight of 10,000 or less or, preferably, that of 500 or lesshas strong anticancer, apoptosis-inducing and antibacterial activities.Thus, depending upon the object, the molecular weight fractionatedfraction of the heat-treated product of the present invention can beused as an effective component of the heat-treated product of thepresent invention.

The heat-treated product of the present invention has an inhibitingactivity to the growth of cancer cells. The action mechanism of theheat-treated product of the present invention does not limit the presentinvention at all and, for example, an apoptosis-inducing action tocancer cells is included in the coverage of the present invention.

The heat-treated product of the present invention has agrowth-inhibiting action and apoptosis-inducing action to cancer cellssuch as human promyelocytic leukemia cells (HL-60), human acutelymphoblastic leukemia cells (MOLT-3), pulmonary cancer cells (A-549),SV40 transformed lung cells (WI-38VA13), hepatic cancer cells (Hep G2),colon cancer cells (HCT 116), human colon cancer cells (SW 480), humancolon cancer cells (WiDr), gastric cancer cells (AGS) and myeloma cellsand the amount of the anticancer substance in the heat-treated productof the present invention can be expressed in terms of an anticanceractivity unit.

The anticancer activity unit used in the present specification isdefined as follows. Thus, the heat-treated solution of the presentinvention is used as a sample, 0.5 ml of its diluted solution is addedto 4.5 ml of an RPMI 1640 medium containing 10% of fetal calf serum and2.5×10⁵ human promyelocytic leukemia cells (HL-60) (ATCC CCL-240),incubated in the presence of 5% carbon dioxide gas at 37° C. for 24hours, numbers of the living cells are counted and the anticanceractivity per ml of the medium when the cell survival rate is 50% of thecontrol is defined as-one unit. Thus, when the anticancer activity perml of the medium is calculated as one unit, then 1 ml of the sample has10 units of anticancer activity.

Survival rate (R) of the cell in terms of % is calculated by thefollowing formula.

R=Vs/(Vs+Ds)×100+Dc/(Vc+Dc)×100

In the formula, Vs and Ds are numbers of viable cells and dead cells,respectively, in the section where the sample has been added; and Vc andDc are numbers of viable and dead cells, respectively, in the sectionwhere water has been added.

The heat-treated product of the present invention is a substance derivedfrom natural food and no toxicity is observed upon oral and parenteraladministrations to mice.

There is no particular limitation for the food and the beverage of thepresent invention and their examples are processed agricultural andforest products, processed livestock products, processed marineproducts, and the like, such as confectionery, bread, noodles, beverages(both alcoholic and nonalcoholic), seasonings, brewing products (soybeanpaste, soybean sauce and vinegar), alcoholic drinks and spicesmanufactured from the raw materials such as cereals, potato, starch,sweeteners, fat/oil, seeds, beans, fish/shellfish, meat of animals,birds and whales, eggs, milks, vegetables, fruits, mushrooms, algae, andthe like.

There is no particular limitation for the methods of manufacturing thefood or the beverage of the present invention and their examples arecooking, processing and commonly-used manufacturing methods for food andbeverages. Any method may be used so far as the manufactured food orbeverage contains the heat-treated product of the present invention.

In the cause of cooking and processing, any method may be used so far asthe product after cooking or processing contains the heat-treatedproduct of the present invention having anticancer action,apoptosis-inducing action, and the like.

Thus, the heat-treated product of the present invention may be addedbefore, during or after cooking or processing. Alternatively, the cookedor processed product of a material thereof may be added to theheat-treated product of the present invention having anticancer action,apoptosis-inducing action, and the like, whereby said heat-treatedproduct is diluted.

Then, in the manufacture of food or beverage, a heating treatment may beconducted in any desired step so that the heat-treated product of thepresent invention having anticancer action, apoptosis-inducing action,and the like, is made contained therein; the heat-treated product of thepresent invention having anticancer action, apoptosis-inducing action,and the like, may be added thereto; or food, beverage or a materialthereof may be added to the heat-treated product of the presentinvention having anticancer action, apoptosis-inducing action, and thelike, so that said heat-treated product is diluted. Addition may beconducted at a time or dividedly in several times. Therefore, it ispossible to easily manufacture a novel food or beverage havinganticancer action, apoptosis-inducing action, and the like. The presentinvention also covers the food or beverage wherein uronic acid, uronicacid lactone, uronic acid ester, a saccharide compound containing uronicacid and/or uronic acid ester or a substance containing such asaccharide compound is made contained during its manufacture so that thefood or beverage is made to consist of its heat-treated product havinganticancer action, apoptosis-inducing action, and the like, producedduring the manufacture. When the product is manufactured by any of thosesteps, the food or beverage containing the heat-treated product of thepresent invention having anticancer action, apoptosis-inducing action,and the like, and those prepared by adding and/or diluting theheat-treated product of the present invention are defined as the food orthe beverage of the present invention.

There is no particular limitation for the content of the heat-treatedproduct of the present invention having anticancer action,apoptosis-inducing action, antibacterial action, and the like but may besuitably chosen in view of its organoleptic property and physiologicalactivity. For example, however, the content of the heat-treated productin 100 parts of food is 0.001 part or more in terms of the heat-treatedproduct of a solid state and, in view of organoleptic property as food,physiological activity such as anticancer action, apoptosis-inducingaction and antibacterial action and the cost, the content is preferably0.005-10 parts or, more preferably, 0.0 1-1part.

There is no particular limitation for the amount of the heat-treatedproduct of the present invention having anticancer action,apoptosis-inducing action, antibacterial action, and the like in thebeverage and may be suitably selected in terms of its organolepticproperty and physiological activity. For example, however; the contentof the heat-treated product in 100 parts of the beverage is 0.001 partor more in terms of the heat-treated product of a solid state and, inview of taste as beverage, physiological activity such as anticanceraction, apoptosis-inducing action and antibacterial action and the cost,the content is preferably 0.005-10 parts or, more preferably, 0.01-1part. Incidentally, the part means that by weight in the presentspecification.

Although the amount of the heat-treated product in the food of thepresent invention having an anticancer action may be suitably selectedin view of the anticancer activity, the amount per 100 g of the food is0.1 unit or more in terms of the anticancer activity unit, preferably 10units or more or, more preferably, 100 units or more.

Although there is no particular limitation for the amount of theheat-treated product having an anticancer action of the presentinvention but the amount may be suitably selected in view of theanticancer activity, the amount per 100 g of the beverage is 0.1 unit ormore in terms of the anticancer activity unit, preferably 10 units ormore or, more preferably, 100 units or more.

There is no particular limitation for the shape of the food or thebeverage of the present invention so far as the heat-treated product ofthe present invention having anticancer action, apoptosis-inducingaction, antibacterial action, and the like, is contained therein, addedthereto and/or diluted therein and the shapes which can be orally takensuch as tablets, granules, capsules, gel, sol, and the like, areadopted.

The food or beverage of the present invention contains the heat-treatedproduct of the present invention having a physiological activity in alarge amount and is a healthy or a functional food or beverageexhibiting carcinogenesis preventing effect, cancer suppressing effect,antiulcer effect, liver function improving effect, constipationpreventing effect, preventing effect for cold by influenza virus andpreventing effect for Alzheimer's disease due to various physiologicalactivities of said heat-treated product such as antibacterial action,apoptosis-inducing action, anticancer action, antiviral action,antiulcer action, antiangiogenic action, liver function improvingaction, dietary fiber action, action of removing unnecessary metals suchas iron and heavy metals, and the like. The food or beverage isparticularly useful for keeping stomach and intestine healthy. Inaddition, it is a food or beverage having a very good preservabilitybecause of its antibacterial action.

The heat-treated product of the present invention may be used as anantiseptic agent for improving the preservability of food or beverage.In addition, the heat-treated product of the present invention may beused in a method for making food or beverage antiseptic by adding it tofood or beverage.

The heat-treated product of the present invention having anantibacterial action can be easily prepared by heating uronic acid,uronic acid lactone, uronic acid ester, a saccharide compound containinguronic acid and/or a saccharide compound containing uronic acid ester,and the like, and the use of the antibacterial agent containing theheat-treated product of the present invention derived from natural foodto food or beverage is quite excellent in terms of safety.

The form of the antibacterial agent containing the heat-treated productof the present invention upon its addition to food or beverage may beany of liquid, paste, powder, flakes, granules, and the like. When aneasy operation or the use by mixing with other additives are taken intoconsideration, it is preferred to make the agent powdery, flaky orgranular by drying. With regard to the method for drying, commonly-usedone such as spray-drying, drum drying, shelf drying, vacuum drying,freeze-drying, and the like, may be used.

The antibacterial agent and antiseptic agent of the present inventionmay be manufactured by any of methods which are known to the personsskilled in the art. Upon the manufacture, known additives which arepermissible for preparing a formulation such as bulking agents,stabilizers, disintegrating agents, binders, auxiliary solubilizers, andthe like, may be appropriately added. Other antibacterial substancessuch as ethanol, glycine, sodium acetate, ascorbic acid, glycerol fattyacid esters, salt, EDTA, and the like, may be jointly used therewith.

Amount of the heat-treated product of the present invention to be addedto food or beverage may vary depending upon the type of the food orbeverage and the amount meeting with the object may be added.

One method of using the antibacterial agent of the present invention isthat where the agent is added to food or to beverage by an appropriatemethod. There is no particular limitation for a method of addition butthat will do ultimately if the heat-treated product of the presentinvention is contained in food or beverage by any means. Accordingly, inthe use of the antibacterial agent of the present invention, the term“addition” covers all methods whereby the heat-treated product of thepresent invention is made contained in the food or beverage. Althoughthe common method is to add it during the manufacturing steps of thefood or beverage, a method where the food is dipped in a solutioncontaining the heat-treated product of the present invention may be usedas well. It is also possible to conduct a method of adding it to thefood together with a method of dipping the food in the solution.Examples of the food which is suitable for a dipping method are the foodwhich does not lose its shape even in water such as fish or livestockmeat paste (e.g., kamaboko [boiled fish paste] and Vienna sausage),noodles (e.g., boiled noodle) and frozen product of fish, shellfish andshrimp before freezing.

When the antibacterial agent of the present invention is used as anantiseptic agent, preservability of food or beverage can be furtherimproved. In the case of frozen food and frozen desert, growth ofcontaminated microorganisms in the processing step before freezing canbe suppressed whereby a very favorable result in terms of hygiene can beobtained. The antibacterial agent of the present invention is effectiveto both gram-positive and gram-negative bacteria and is very effective,for example, to drug-resistant bacteria such as methicillin-resistantStaphylococcus aureus and bacteria which cause food poisoning such asSalmonella, enterotoxin-producing Staphylococcus aureus, Bacillus cereusof a vomiting type, Bacillus cereus of a diarrhea type andenterorrhagial Escherichia coli O-157. Said agent is effective tomicroorganisms such as yeasts and fungi as well. The antiseptic agentcontaining the heat-treated product of the present invention isparticularly and highly useful as a natural preventive agent for foodpoisoning and as a sterilizing agent. Incidentally, sterilization ofclothing, bed sheet, and the like, can be conducted using theantibacterial agent of the present invention and, when the antibacterialagent of the present invention is sprinkled or when wiping-off with theantibacterial agent of the present invention is conducted, it ispossible to sterilize (both to remove and to kill the bacteria) theobject to be sterilized.

The antibacterial agent of the present invention shows an antibacterialactivity to bacteria for dental caries, and those for periodontaldisease and an intraoral preparations containing the antibacterial agentof the present invention can be offered. The form of the intraoralpreparation may be a known one such as liquid or paste. An example ofthe intraoral preparation is a dentifrice. The dentifrice may be in aknown form such as liquid, paste or powder. There is no particularlimitation for the amount of the heat-treated product of the presentinvention in the dentifrice and, if an effective concentration to thebacteria for dental caries and for periodontal disease is containedtherein, that will be enough. Known additives such as moisturizingagents, surface-active agents, binders, perfumes, sweetening agents, andthe like, may be added to the dentifrice. As mentioned already,heat-treated product of the substances which contain a saccharidecompound containing uronic acid or uronic acid ester such aspectin-containing substance (e.g. vegetables and fruits) may be used aswell and an intraoral preparation containing a heat-treated product ofpectin-containing vegetable such as dentifrice may be included in thecoverage of the present invention.

To prepare the apoptosis inducer of the present invention, theheat-treated product of the present invention having anapoptosis-inducing ability is employed as the active ingredient andcompounded with known pharmaceutical carriers to give a pharmaceuticalpreparation. Usually, the heat-treated product of the present inventionis compounded with pharmaceutically acceptable liquid or solid carriersfollowed, if necessary, by adding solvents, dispersing agents,emulsifiers, buffers, stabilizers, bulking agents, binders,disintegrating agents, lubricants and the like thereto whereupon solidpreparations such as tablets, granules, diluted powders, powders,capsules, and the like, or liquid preparations such as solutions,suspensions, emulsions, and the like, are prepared. The resultingpreparation may be processed into a dry one which can be then liquefiedprior to use by adding an appropriate carrier thereto.

The apoptosis inducer of the present invention can be administeredeither orally or parenterally by, for example, injection or intravenousdrip infusion.

The pharmaceutical carriers may be appropriately selected depending uponthe administration route and dosage form as mentioned above. Starch,lactose, sucrose, mannitol, carboxymethylcellulose, corn starch,inorganic salts and the like may be used in the case of the oralpreparations. In preparing the oral preparations, it is also possible toadd binders, disintegrating agents, surface-active agents, lubricants,fluidity improving agents, corrigents, coloring agents, perfumes and thelike thereto.

On the other hand, in the case of parenteral preparations, theheat-treated product having an apoptosis inducing activity which is anactive ingredient of the present invention is dissolved or suspended bya common manner in a diluent such as distilled water for injection,physiological saline solution, aqueous solution of glucose, plant oilfor injection, sesame oil, peanut oil, soybean oil, corn oil, propyleneglycol or polyethylene glycol followed, if necessary, by addingbactericides, stabilizers, isotonic agent, analgesic agents, and thelike thereto whereupon the desired parenteral preparation is obtained.

The apoptosis inducer of the present invention is administered via anappropriate administration route depending upon the dosage form. Thereis no particular limitation for the method of administration as well andany of internal and external route and a route by injection may beselected therefor. Injections may be administered, for example, byintravenous, intramuscular, subcutaneous and intradermal routes whilepreparations for external use include suppositories.

The dose of the apoptosis inducers of the present invention is notparticularly specified but may be appropriately determined dependingupon the dosage form, administration method, purpose of the use and theage, body weight, conditions, and the like, of the patient to whom theinducer is administered. Usually, however, the dose of the heat-treatedproduct of the present invention contained in the preparation for anadult is 20-2,000 mg/kg per day. As a matter of course, the dose mayvary depending upon various factors and, therefore, less dose than theabove-mentioned one may be sufficient in some cases while, in othercases, more dose than the above may be necessary. The agent of thepresent invention may be administered orally as it is and, further, theagent may be taken daily after adding to common food and/or beverage aswell.

An anticancer agent can be manufactured when the heat-treated product ofthe present invention having an anticancer action is used as an activeingredient and is made into a pharmaceutical preparation together withknown pharmaceutical carriers. The anticancer agent may be manufacturedin accordance with the method mentioned above. Usually, the heat-treatedproduct of the present invention is compounded with pharmaceuticallyacceptable liquid or solid carriers followed, if necessary, by addingsolvents, dispersing agents, emulsifiers, buffers, stabilizers, bulkingagents, binders, disintegrating agents, lubricants, and the like, togive solid preparations such as tablets, granules, diluted powders,powders, capsules, and the like, or liquid preparations such assolutions, suspensions, emulsions, and the like. Alternatively, it maybe processed into a dry preparation which can be liquefied by adding anappropriate carrier thereto before actual use.

The anticancer agent of the present invention may be administered eitherorally or parenterally by, for example, means of injection orintravenous drip infusion.

The pharmaceutical carriers may be appropriately selected depending uponthe above-mentioned administration route and dosage form and may be usedin the same manner as in the case of the apoptosis inducer mentionedalready.

The anticancer agent is administered by an appropriate administrationroute depending upon the dosage form. There is no particular limitationfor the administration method and, for example, administration byinternal or external route or by injection may be conducted. Injectionsmay be administered, for example, by intravenous,intramuscular,subcutaneous and intradermal routes while preparations forexternal use include suppositories.

The dose of the anticancer agent of the present invention is notparticularly specified but may be appropriately determined dependingupon the dosage form, administration method, purpose of the use and theage, body weight, conditions, and the like, of the patient to whom theagent is administered. Usually, however, the dose of the heat-treatedproduct of the present invention contained in the preparation for adultsis 20-2,000 mg/kg per day. As a matter of course, the dose may varydepending upon various factors and, therefore, less dose than theabove-mentioned one may be sufficient in some cases while, in othercases, more dose than the above may be necessary. The agent of thepresent invention may be administered orally as it is and, further, theagent may be taken daily after adding to common food and/or beverage aswell.

The heat-treated product of the present invention has an anticanceraction and, at low concentrations, it has an ability of inducing adifferentiation of cancer cells whereby the heat-treated product of thepresent invention is also useful as a differentiation-inducing agent(decarcinogenic agent). The differentiation inducer for cancer cellscontaining the heat-treated product of the present invention as anactive ingredient can be made into preparations by the same manner inthe case of the anticancer agent mentioned above and can be administeredby the same method as that in the case of the anticancer agent.

The dose of the agent as a differentiation inducer for cancer cells isnot particularly specified but may be appropriately determined dependingupon the dosage form, administration method, purpose of the use and theage, body weight, conditions, and the like, of the patient to whom theinducer is administered. Usually, however, the dose of the heat-treatedproduct of the present invention contained in the preparation for anadult is 0.2-500 mg/kg per day. As a matter of course, the dose may varydepending upon various factors and, therefore, less dose than theabove-mentioned one may be sufficient in some cases while, in othercases, more dose than the above may be necessary. The agent of thepresent invention may be administered orally as it is and, further, theagent may be taken daily after adding to common food and/or beverage aswell.

The heat-treated product of the present invention has an antiviraleffect and an action of improving the hepatic function. Accordingly,antiviral agent and a hepatic function improving agent containing theheat-treated product of the present invention as an active ingredientcan be prepared by the same manner as in the case of the above-mentionedanticancer agent and can be administered by the same manner as in thecase of the anticancer agent.

The dose as the antiviral agent and the hepatic function improving agentis not particularly specified but may be appropriately determineddepending upon the dosage form, administration method, purpose of theuse and the age, body weight, conditions, and the like, of the patientto whom the agent is administered. Usually, however, the dose of theheat-treated product of the present invention contained in thepreparation for adults is 0.2-2,000 mg/kg per day. As a matter ofcourse, the dose may vary depending upon various factors and, therefore,less dose than the above-mentioned one may be sufficient in some caseswhile, in other cases, more dose than the above may be necessary. Theagent of the present invention may be administered orally as it is and,further, the agent may be taken daily after adding to common food and/orbeverage as well. When the preparation containing the heat-treatedproduct of the present invention is administered, viral diseases such ascommon cold caused by influenza virus can be prevented and treated and,in addition, hepatic function disorder can be improved as well wherebyGOT and GPT values become normal.

The heat-treated product of the present invention has an action ofinducing a heat shock protein of, for example, 70-k daltons and exhibitsan antiviral action to RNA viruses and DNA viruses such as hepatitisvirus, AIDS virus, influenza virus, herpes virus, and the like. It showsa bioprotective action such as an antiinflammatory action.

An antiulcer agent can be prepared by using the heat-treated product ofthe present invention having an antiulcer action as the activeingredient together with known pharmaceutical carriers followed byprocessing into a-pharmaceutical preparation. The antiulcer agent can beprepared in accordance with the method described above. Usually, theheat-treated product of the present invention is compounded withpharmaceutically acceptable liquid or solid carriers followed, ifnecessary, by adding solvents, dispersing agents, emulsifiers, buffers,stabilizers, bulking agents, binders, disintegrating agents, lubricants,and the like thereto whereby solid preparations such as tablets,granules, diluted powders, powders, capsules, and the like, or a liquidpreparations such as solutions, suspensions, emulsions, and the like,are prepared. It is also possible to prepare a dry product which can bemade into liquid by addition of an appropriate carrier before use.

The antiulcer agent may be administered by an oral route or by aparenteral route as injections or intravenous drip infusion.

The pharmaceutical carrier may be selected depending upon theabove-mentioned administration manner and dosage form and may be used bythe same manner as in the case of the above-mentioned apoptosis inducer.

The antiulcer agent may be administered via an appropriateadministration route depending upon the dosage form. The administrationmethod is not particularly limited too and administration by internal orexternal route or by injection may be conducted. Injections may beadministered, for example, by intravenous, intramuscular, subcutaneousor intradermal route. Preparations for external use includesuppositories.

The dose as the antiulcer agent is not particularly specified but may beappropriately determined depending upon the dosage form, administrationmethod, purpose of the use and the age, body weight, conditions, and thelike, of the patient to whom the agent is administered. Usually,however, the dose of the heat-treated product of the present inventioncontained in the preparation for an adult is 20-2,000 mg/kg per day. Asa matter of course, the dose may vary depending upon various factorsand, therefore, less dose than the above-mentioned one may be sufficientin some cases while, in other cases, more dose than the above may benecessary. The agent of the present invention may be administered orallyas it is and, further, the agent may be taken daily after adding tocommon food and/or beverage as well.

The present invention offers food or beverage which has a physiologicalactivity such as anticancer action and apoptosis-inducing action,induces anticancer action or apoptosis in ill cells in the patientssuffering from cancer or viral diseases and is effective for preventionand therapy of said disease. Especially in the case of cancer ofdigestive organs such as cancer of stomach and colon, it is possible toinhibit the growth of cancer cells or to result in apoptosis in cancercells by giving the heat-treated product of the present invention byoral route as food or beverage and, therefore, the food or beveragewhere the heat-treated product of the present invention is containedtherein, added thereto and/or diluted therein has an excellent effectfor therapy and prevention of cancers of digestive organs.

In addition, the heat-treated product of the present invention hasantiviral and antibacterial actions. Therefore, it is useful asantiviral agent, antibacterial agent, intraoral agent (such asdentifrice) and antiseptic agent for food or beverage and, due to itsantiulcer action, it is also useful as antiulcer agent and a preventiveagent for ulcer. Further due to its action for improving the hepaticfunction, it is useful as a hepatic function improving agent too.

It is now possible in accordance with the present invention that thefood or beverage of the present invention contains a large amount of theheat-treated product of the present invention having a physiologicalactivity. The food or beverage of the present invention is a healthy orfunctional food or beverage exhibiting a maintenance action ofhomeostasis of living body such as carcinogenesis preventing effect,anticancer effect, antibacterial effect, antiviral effect, antiulcereffect, constipation preventing effect, hepatic function improvingeffect, preventing effect for Alzheimer disease, apoptosis-inducingeffect, and the like, due to various physiological activities of saidheat-treated product such as apoptosis-inducing action, antibacterialaction, anticancer action, antiviral action, antiangiogenic actioninhibitory, action for abnormally proliferating cells, antiulcer action,hepatic function improving action, dietary fiber action, action ofremoving unnecessary metals such as iron and heavy metals, and the like.Thus, in accordance with the present invention, food or beveragecontaining functional substances which is useful for keeping stomach andintestine healthy. When the heat-treated product of the presentinvention, especially a fraction having a molecular weight of 500 orless, is added, the antibacterial activity of food and beverage can beeasily made strong and, therefore, the heat-treated product of thepresent invention is quite useful as an antiseptic agent for food andbeverage as well. Due to its various physiological functions, when theheat-treated product of the present invention (particularly a fractionhaving a molecular weight of 10,000 or less or, preferably, that havinga molecular weight of 500 or less) is used in food or beverage, it isnow possible to easily give various physiological functions to food orbeverage. Thus, the heat-treated product is quite useful, for example,as an antibacterial additive to food or beverage and also as anantiseptic agent for food or beverage.

The present invention further offers an apoptosis inducer and ananticancer agent which are useful for prevention and therapy of patientssuffering from cancer and viral diseases by inhibiting the proliferationof pathogenic cells and by inducing apoptosis to pathogenic cells due toits anticancer and apoptosis-inducing actions. Especially in the case ofcancer of digestive organs such as cancer of stomach and colon, it ispossible to inhibit the growth of cancer cells or to result in apoptosisin cancer cells by administering the heat-treated product of the presentinvention by oral route as food or beverage and, therefore, the food orbeverage where the heat-treated product of the present invention iscontained therein, added thereto and/or diluted therein has an excellenteffect for therapy and prevention of cancers of digestive organs. Thepresent invention furthermore offers an antiulcer agent having anantiulcer action which is useful for prevention and therapy of ulcer forthe patients suffering from said disease. In the case of ulcer ofdigestive organs, the heat-treated product of the present inventionachieves an antiulcerative action by taking it orally as food orbeverage and, therefore, the food or beverage where the heat-treatedproduct of the present invention is added thereto and/or diluted thereinhas an excellent effect for therapy and prevention of ulcers ofdigestive organs. The pharmaceutical agent of the present invention canbe supplied in low cost and in large quantities using edible fruit rind,edible algae, and the like as a starting material and another advantageis that it has a high safety because it is derived from food. Moreover,a simple method for inducing apoptosis can be offered by the presentinvention and, when the method of the present invention is used, it isnow possible to study for clarifying the mechanism of apoptosis and todevelop inhibitors to an apoptosis induction.

EXAMPLES

The present invention will be further illustrated by way of thefollowing examples although the present invention is never limited byand to those examples. Incidentally, the term % used in the examplesmeans that by weight.

Example 1

Pectin which was manufactured from apple (manufactured by Wako PureChemicals) (500 mg) was suspended in 50 ml of 50 mM HEPES buffer (pH:7.0) containing 120 mM of NaCl and autoclaved at 121° C. for 20 minutesto prepare a heat-treated pectin solution.

Human promyelocytic leukemia cells HL-60 (ATCC CRL-1964) were incubatedin an RPMI 1640 medium (manufactured by Nissui) containing 10% of fetalcalf serum (manufactured by Gibco) treated at 56° C. for 30 minutes andthen suspended in an ASF 104 medium (manufactured by Ajinomoto) to makethe cell concentration 5×10⁵cells/9 ml.

To this suspension was added 1 ml of the heat-treated pectin solutionand the mixture was incubated at 37° C. for 16 hours in the presence of5% of carbon dioxide. For the sake of confirmation, the same incubationas above was conducted except that 0.1 ml of aqueous solution (0.1mg/ml) of actinomycin D (manufactured by Sigma) which was known as anapoptosis-inducing reagent and 0.9 ml of a physiological saline solutionwere used instead of the above-mentioned pectin solution.

The incubated cells were observed under an optical microscope whereuponcondensation of nuclei, contraction of cells and production of apoptoticbody were confirmed in both of the heat-treated pectin solution and theactinomycin D-added incubated cells. Incidentally, in the control wherethe cells to which 1 ml of physiological saline solution was added wereincubated, such phenomena were not observed.

From those results, it was found that the heat-treated pectin solutioninduced apoptosis in HL-60 cells.

Example 2

Commercially available pectin manufactured from apple was dissolved in a50 mM HEPES buffer (pH: 7.0) containing 120 mM of NaCl so as to make thefinal concentration of the pectin 10 mg/ml and then the solution wasadjusted to pH 7.0 with 1N NaOH. This was heated at 121° C. for 30minutes and its ultraviolet absorption spectrum was measured whereuponthe absorbance at around 235 nm of the heat-treated product increased ascompared with that before heating.

This sample was adjusted to pH 7.0 with 1N NaOH and theapoptosis-inducing activity was measured by a method mentioned inExample 1. In this and all of the succeeding examples, however, therewere some exceptions that an RPMI 1640 medium containing 10% of fetalbovine serum was used instead of an ASF 104 medium, that HL-60 (ATCCCCL-240) was used as the cells and that, upon measurement of theapoptosis-inducing activity, each of the samples was adjusted to pH 7.0with 1N NaOH whereby the apoptosis-inducing activity was measured. Tothe cell suspension was added twice as much by volume of 0.4% aqueoussolution of trypan blue and an observation was conducted under anoptical microscope whereby trypan blue was excreted and colorless cellsand blue-colored cells were counted as viable and dead cells,respectively.

As a result thereof, the heat-treated pectin product showed asignificant apoptosis-inducing activity to HL-60 cells.

Commercially available pectin from lemon was dissolved in 50 mM HEPESbuffer (pH: 7.0) containing 120 mM of NaCl to make the concentration ofthe pectin 10 mg/ml whereupon the pH was 5.0. This was heated at 121° C.for 30 minutes and an ultraviolet absorption spectrum was measuredwhereupon the absorbance at around 235 nm increased in the heat-treatedproduct.

This sample was adjusted to pH 7.0 with 1N NaOH and, when theapoptosis-inducing activity to HL-60 cells was measured by theabove-mentioned method, the heat-treated product was found to exhibit asignificant apoptosis-inducing activity.

The results are shown in FIG. 1. Thus FIG. 1 shows a relationshipbetween the incubation time and the viable cell number in the culturemedium when a heat-treated lemon pectin solution was added to a culturemedium of HL-60 cells to make the pectin concentration 1 mg/ml whereinthe abscissa is the incubation time (hours) while the ordinate is theviable cell number (×10⁵ cells/5 ml) in the culture medium. In FIG. 1,the open square stands for the control where no sample was added whilethe open rhombus stands for the case where heat-treated lemon pectin wasadded. Thus, the heat-treated lemon pectin showed an anticancer action.

Example 3

(1) Commercially available pectin manufactured from apple was dissolvedin 50 mM HEPES buffer (pH: 7.0) containing 120 MM of NaCl to make thepectin concentration 10 mg/ml and heated at 121° C. for 20 minutes toprepare a heat-treated solution. A part of it was freeze-dried to give aheat-treated solution made into a freeze-dried state.

Then the remaining part of the heat-treated solution was dialyzedagainst pure water using a Seamless cellulose tubing (cutoff molecularweight: 12,000-14,000; manufactured by Sanko Junyaku) or Spectra/Por 7dialyzing membrane (cutoff molecular weight: 1,000; manufactured bySpectrum) and each of the inner liquids after dialysis was freeze-driedand weighed whereupon, in each of the freeze-dried inner liquids, therewas a loss in weight of about 10% as compared with the pectin before theheating treatment.

The freeze-dried heat-treated solution was dissolved in water while thefreeze-dried inner liquid after the dialysis was dissolved in 50 mMHEPES buffer (pH: 7.0) containing 120 mM of NaCl whereupon the finalconcentration of each of the both solutions was made 10 mg/ml. Thesolution was adjusted to pH 7.0 with 1NaOH and an apoptosis-inducingactivity to HL-60 cells was measured by the method as mentioned inExample 2.

The results were that the heat-treated pectin solution showed anactivity while the inner liquid after the dialysis showed a decreasedactivity.

The results are shown in FIG. 2. Thus, FIG. 2 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when a freeze-dried heat-treated solution, a freeze-dried innerliquid after dialyzed using a cellulose membrane or a freeze-dried innerliquid after dialyzed using Spectra/Por 7 dialyzing membrane was addedto a culture medium of HL-60 cells to make the concentration 1 mg/mlwherein the abscissa stands for the incubation time (hours) while theordinate stands for the viable cell number (×10⁵ cells/5 ml) in theculture medium. In FIG. 2, open square stands for the control where nosample was added; open rhombus stands for the case where thefreeze-dried product of the heat-treated solution was added; open circlestands for the case where freeze-dried product of the inner liquid afterthe dialysis through cellulose membrane was added; and open trianglestands for the case where freeze-dried product of the inner liquid afterthe dialysis through the Spectra/Por 7 dialyzing membrane was used.Thus, the heat-treated solution exhibited an anticancer action.

(2) After the above-mentioned heat-treated pectin solution was adjustedto pH 7.0 with 1N NaOH and subjected to an ultrafiltration using aCentriplus 10 (fractionating molecular weight: 10,000; manufactured byAmicon) to prepare a fraction which passed through the membrane. Theapoptosis-inducing activity of this fraction was measured by a methodmentioned in Example 2 whereupon it had the same activity as the samplebefore the ultrafiltration had.

The results are shown in FIG. 3. Thus, FIG. 3 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when a fraction of the heat-treated pectin solution passingthrough Centriplus 10 was added to a culture medium of HL-60 cells tomake the concentration 1 mg/ml wherein the abscissa stands for theincubation time (hours) while the ordinate stands for the viable cellnumber (×10⁵ cells/5 ml) in the culture medium. In FIG. 3, open squarestands for the control where no sample was added and open rhombus standsfor the case where the fraction passing through the membrane was added.Thus, the heat-treated pectin solution exhibited the same result as thecase of open rhombus and the heat-treated pectin solution and thefraction passing through the membrane showed an anticancer action.

Example 4

Commercially available pectin manufactured from apple was dissolved in50 mM HEPES buffer (pH: 7.0) containing 120 mM of NaCl to make thepectin concentration 10 mg/ml and the solution was adjusted to pH 7.0with 1N NaOH and heated at 121° C. for 30 minutes. This sample (20 ml)was applied to a column of Sephacryl S-300 Hiload 26/60 High Resolution(manufactured by Pharmacia) equilibrated with pure water and subjectedto gel filtration. Pure water was used for the mobile phase at the flowrate of 1 ml/minute and detection was performed by a differentialrefractometer.

Each of the fraction 1 (which was eluted after 110-190 minutes fromapplication of the sample to the column), fraction 2 (eluted after190-270 minutes) and fraction 3 (eluted after 270-400 minutes) wasconcentrated by means of an evaporator. To each of the fraction wereadded NaCl and HEPES to make their final concentrations 120 mM and 50mM, respectively and to make the volume 20 ml. This was adjusted to pH7.0 with 1N NaOH.

An apoptosis-inducing activity to HL-60 cells was measured by the methodof Example 2 whereupon a strong activity was found in the fraction 3having the lowest molecular weights.

The results are shown in FIG. 4. Thus, FIG. 4 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when the above-mentioned fraction 3 was added to a culture mediumof HL-60 cells to make the concentration 1 mg/ml wherein the abscissastands for the incubation time (hours) while the ordinate stands for theviable cell number (×10⁵ cells/5 ml) in the culture medium. In FIG. 4,open square stands for the control where no sample was added and opentriangle stands for the case where the fraction 3 was added. Thus, thefraction 3 exhibited an anticancer action.

Example 5

D-α-galacturonic acid or D-glucuronic acid were dissolved in 50 mM HEPESbuffer (pH: 7.0) containing 120 mM of NaCl to make the concentration ofthe acids 10 mg/ml. The resulting solutions were heated at 121° C. for20 minutes and adjusted to pH 7.0 with 1N NaOH. The apoptosis-inducingactivity of those samples to HL-60 cells was measured by the method ofExample 2 whereupon both samples exhibited significant activity.

The results are shown in FIG. 5. Thus, FIG. 5 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when the heat-treated galacturonic acid solution or theheat-treated glucuronic acid were added to a culture medium of HL-60cells to make the concentration of the acids 1 mg/ml wherein theabscissa stands for the incubation time (hours) while the ordinatestands for the viable cell number (×10⁵ cells/5 ml) in the culturemedium. In FIG. 5, open square stands for the control where no samplewas added, open rhombus stands for the case where the heat-treatedgalacturonic acid was added and open circle stands for the case wherethe heat-treated glucuronic acid was added. Thus, both of theheat-treated products exhibited an anticancer action.

(2) Galacturonic acid was dissolved in 50 mM HEPES buffer (pH: 7.0)containing 120 mM of NaCl to make the acid concentration 10 mg/ml. Thesolution was adjusted to pH 7.0 and to pH 8.0 with 1N NaOH. Each of themwas heated at 121 ° C. for 20 minutes and then adjusted to pH 7.0 with1N NaOH. Apoptosis inducing activity of those samples to HL-60 cells wasmeasured by the method of Example 2 whereupon the sample heated at pH7.0 showed stronger activity than that heated at pH 8.0.

The results are shown in FIG. 6. Thus, FIG. 6 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when the heat-treated solutions of galacturonic acid at pH 7.0 or8.0 were added to make the concentration 1 mg/ml wherein the abscissastands for the incubation time (hours) while the ordinate stands for theviable cell number (×10⁵ cells/5 ml) in the culture medium. In FIG. 6,open square stands for the control where no sample was added, openrhombus stands for the case where the galacturonic acid heated at pH 7.0was added and open circle stands for the case where the galacturonicacid heated at pH 8.0 was added. Thus, the product heated at pH 7.0showed an anticancer activity.

Example 6

Pectin manufactured from apple was dissolved in 50 mM HEPES buffer (pH:7.0) containing 120 mM of NaCl to make the pectin concentration 10 mg/mland the solution was heated at 121° C. for 20 minutes to give aheat-treated sample 1. This was dialyzed against 50 mM HEPES buffer (pH:7.0) containing 120 mM of NaCl using above-mentioned cellulose dialyzingmembrane to prepare an inner liquid sample 2. The inner liquid sample 2after the dialysis was further heated at 121 ° C. for one hour followedby adjusting to pH 7.0 with1N NaOH to prepare a re-heated sample 3.

Each of the samples 1-3 were adjusted to pH 7.0 with 1N NaOH and anapoptosis-inducing activity to HL-60 cells of them was measured by themethod of Example 2 whereupon it was found that the samples 1 and 3showed the activity while, in the case of the sample 2, the activitydecreased.

It is clear from those results that the inner liquid of the heat-treatedpectin after dialysis having a decreased activity due to the dialysisrecovers its activity by means of the re-heating.

Example 7

Commercially available pectin manufactured from apple was dissolved in1NHCl to make the pectin concentration 10 mg/ml and the solution washeated at 121° C. for 1.5 hours to prepare a heat-treated product. Thensaid heat-treated product was adjusted to pH 7.0 with NaOH and itsapoptosis-inducing activity to human promyelocytic leukemia cells(HL-60) was measured as follows.

Thus, HL-60 (ATCC CCL-240) were incubated in an RPMI 1640 medium(manufactured by Nissui) containing 10% of fetal calf serum(manufactured by Gibco) treated at 56° C. for 30 minutes and thensuspended in an RPMI 1640 medium to make the cell concentration 2.5×10⁵cells/4.5 ml.

To 4.5 ml of this suspension was added 0.5 ml of the above-mentionedheat-treated pectin solution and the mixture was incubated at 37° C. for16 hours in the presence of 5% of carbon dioxide. For the sake ofconfirmation, the same incubation as above was conducted except that0.05 ml of an aqueous solution (0.1 mg/ml) of actinomycin D(manufactured by Sigma) which was known as an apoptosis-inducing reagentand 0.45 ml of a physiological saline solution were used instead of theabove-mentioned heat-treated pectin solution.

The incubated cells were observed under an optical microscope whereuponcondensation of nuclei, contraction of cells and production of apoptoticbody were confirmed in both of the heat-treated pectin solution and theactinomycin D-added incubated cells. Incidentally, in the control wherethe cells to which 0.5 ml of a physiological saline solution was addedwere incubated, such phenomena were not observed.

Further, to the cell suspension was added twice as much by volume of a0.4% aqueous solution of trypan blue and an observation was conductedunder an optical microscope whereby trypan blue was excreted andcolorless cells and blue-colored cells were counted as viable and deadcells, respectively.

The results are shown in FIG. 7. Thus, FIG. 7 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when the heat-treated pectin solution was added to the culturemedium of HL-60 cells to make the pectin concentration 1 mg/ml whereinthe abscissa stands for the incubation time (hours) while the ordinatestands for the viable cell number (×10⁵ cells/S ml) in the culturemedium. In FIG. 7, open square stands for the control where no samplewas added and open rhombus stands for the case where the heat-treatedpectin solution was added. Thus, the heat-treated pectin showed ananticancer activity.

Example 8

Commercially available pectin manufactured from apple was dissolved inwater to make the pectin concentration 10 mg/ml and the solution wasadjusted to pH 7.0 with NaOH and heated at 121° C. for one hour. The pHafter the heating was 4.5. Then this heat-treated product was adjustedto pH 7.0 with NaOH again, insoluble matters therein were removed bymeans of a centrifugation (10,000×g for ten minutes) and of a filtrationusing a filter of 0.22 μm, then ethanol of the same volume was addedthereto, the mixture was centrifuged (10,000×g for ten minutes), each ofthe resulting supernatant fraction and precipitate fraction wasevaporated to dryness in vacuo and each of them was dissolved in waterof the amount which was same as that used for dissolving the pectin inthe initial stage. Each of the aqueous solutions of the ethanol-treatedsupernatant fraction and of the precipitate fraction was adjusted to pH7.0 with NaOH and 0.5 ml of each of them was added to 4.5 ml of aculture medium of HL-60 cells to measure the apoptosis-inducing activityby the method of Example 7.

As a result thereof, it was found that the apoptosis-inducing activityto HL-60 cells was present in the supernatant fraction. The same resultwas obtained when 2-propanol was used instead of ethanol. The resultsare shown in FIG. 8. Thus, FIG. 8 shows the relationship between theincubation time and the viable cell number in the culture medium whenthe aqueous solution of the supernatant fraction or the precipitatefraction after treating with ethanol or with 2-propanol was added to theculture medium of HL-60 cells wherein the abscissa stands for theincubation time (hours) while the ordinate stands for the viable cellnumber (×10⁵ cells/5 ml) in the culture medium. In FIG. 8, open squarestands for the control where no sample was added, open circle stands forthe case where the ethanol-treated precipitate fraction was added,closed circle stands for the case where the ethanol-treated supernatantfraction was added, open triangle stands for the case where the2-propanol-treated precipitate fraction was added and closed trianglestands for the case where the 2-propanol-treated supernatant fractionwas added. Thus, the solvent-treated supernatant fractions showed ananticancer activity.

Samples were prepared by the same method as mentioned above by changingthe amount of ethanol or 2-propanol to be added to the heat-treatedpectin to 0.5, 1.5 and 2-fold by volume whereupon it was found that,like in the cases where the equivalent volume of ethanol or 2-propanolwas added, the activity was noted in the supernatant fractions.Incidentally, the apoptosis-inducing activity was measured by thefollowing method. Thus, to each of the wells of a 96 well microtiterplate were added 100 microliters of an RPMI 1640 medium containing 10%of fetal bovine serum containing 5,000 HL-60 cells, 10 microliters ofthe sample and 10 microliters of alamarBlue (manufactured by AlamarBioscience) and incubation was conducted at 37° C. for 48 hours in thepresence of 5% of carbon dioxide gas. After that, the value obtained bysubtracting the absorbance at 590 nm from that at 560 nm was measuredand this was defined as a degree of proliferation of the cells.

Example 9

Commercially available pectin manufactured from apple was dissolved in a0.1M carbonate buffer to make the pectin concentration 10 mg/ml and thepH was adjusted to 9.5. This solution was heated at 121° C. for 30minutes. The pH of the heat-treated product was 9.2. Then a part of theheat-treated product was adjusted to pH 7.0 with HCl (sample A) whilethe remainder was adjusted to pH 4.5. The sample adjusted to pH 4.5 washeated again at 121° C. for 30 minutes and the pH was adjusted to pH 7.0(sample B). The apoptosis-inducing activity of the samples A and B toHL-60 cells was measured by the method of Example 7 whereupon it wasfound that sample A did not show the activity while sample B(heat-treated pectin solution II) showed the activity.

The results are shown in FIG. 9. Thus, FIG. 9 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when sample A or B was added to the culture medium of HL-60 cellswherein the abscissa stands for the incubation time (hours) while theordinate stands for the viable cell number (×10⁵ cells/5 ml) in theculture medium. In FIG. 9, open square stands for the control where nosample was added, open rhombus stands for the case where the sample Awas added and open circle stands for the case where the sample B wasadded. Thus, the heat-treated pectin solutions showed the anticanceractivity.

Example 10

(1) When D-α-galacturonic acid was dissolved in water to make theconcentration 10 mg/ml whereupon the pH was 2.4. This was heated at 121°C. for 20 minutes. The pH of the heat-treated product was 2.2. The pH ofthis heat-treated product was adjusted to pH 7.0 and theapoptosis-inducing activity to HL-60 cells was measured by the method ofExample 7 with an exception that the cell suspension in which HL-60 cellnumbers were adjusted to 3×10⁵ cells/4.5 ml was used whereby the presentsample was found to have the activity.

The results are shown in FIG. 10. Thus, FIG. 10 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when the heat-treated product of galacturonic acid under anacidic condition was added to a culture medium of HL-60 cells to makethe concentration 1 mg/ml wherein the abscissa stands for the incubationtime (hours) while the ordinate stands for the viable cell number (×105cells/5 ml) in the culture medium. In FIG. 10, open square stands forthe control where no sample was added and open rhombus stands for thecase where the heat-treated galacturonic acid was added. Thus, theheat-treated product showed the anticancer activity.

(2) D-Glucuronic acid was added to 50 mM HEPES buffer (pH: 7.0)containing 120 mM of NaCl to make the concentration 10 mg/ml whereuponthe pH was 3.18. The solution was heated at 121° C. for 20 minutes, pHof the heat-treated solution was adjusted to 7.0 with NaOH and theapoptosis-inducing activity to HL-60 cells was measured by the method ofExample 7 whereupon the present sample was found to have the activity.

The results are shown in FIG. 11. Thus, FIG. 11 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when heat-treated glucuronic acid was added to the culture mediumof HL-60 cells to make the concentration 1 mg/l ml wherein the abscissastands for the incubation time (hours) while the ordinate stands for theviable cell number (×10⁵ cells/5 ml) in the culture medium. In FIG. 11,open square stands for the control where no sample was added while opencircle stands for the case where the heat-treated glucuronic acid wasadded. Thus, the heat-treated glucuronic acid product showed theanticancer activity.

Example 11

When Dα-galacturonic acid was dissolved in water to make theconcentration 1% whereupon the pH was 2.4. When this solution was heatedat 121° C. for 20 minutes, pH of the heat-treated solution was 2.2. Thiswas concentrated to an extent of 40-fold in vacuo and 20 microliters ofthe concentrate was subjected to a high-performance liquidchromatography using a column of Palpak Type S (4.6×250 mm; manufacturedby Takara Shuzo). Then the galacturonic acid which was heated under anacidic condition was separated therefrom using an aqueous solution ofacetonitrile at the flow rate of 1 ml/minute. During the first 30minutes, a 90% solution was used and, during the succeeding 20 minutes,a linear concentration gradient was applied using 90% to 50% solutions.Fractionation was conducted every 90 seconds, each fraction wasevaporated to dryness in vacuo, then was dissolved in 80 microliters ofwater and each 10 microliters of the solution were subjected to ameasurement of the apoptosis-inducing activity to HL-60 cells by an MTTmethod which will be given below.

As a result thereof, the activity was found in the two fractions havingeluting times of 4.5-12 minutes and 45-48 minutes.

MTT Method: Each of 5 microliters of the diluted solution of each sampleliquid or 5 microliters of water was placed in the well of a 96 wellmicrotiter plate. To it was added 100 microliters of an RPMI 1640 mediumcontaining 10% of fetal bovine serum containing 5,000 HL-60 cells and anincubation was conducted at 37° C. for 48 hours in the presence of 5% ofcarbon dioxide gas. After addition of 10 microliters of phosphatebuffered saline containing 5 mg/ml of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;manufactured by Sigma) hereto, the incubation was conducted for 4 hoursmore and the growth state of the cells was observed under a microscope.On the other hand, 100 microliters of 2-propanol containing 0.04N HClwas added thereto, the mixture was well stirred and the absorbance at590 nm was measured and used as a degree of proliferation of the cells.

Example 12

(1) Commercially available pectin manufactured from apple was suspendedin water to make the pectin concentration 2.5%. The suspension wasadjusted to pH 7.0 with NaOH, placed in a dialyzing tube whosefractionating molecular weight is 12,000-14,000 and dialyzed against15-fold by volume of water for four times. After being dialyzed, thesolution was adjusted to pH 7.0 again and heated at 121° C. for one hourto prepare a heat-treated solution. The pH of this heat-treated solutionwas 5.4. The pH of this heat-treated solution was adjusted to 7.0 withNaOH, subjected to a centrifugation to remove the insoluble matters andsubjected to filtration using the filters of 0.8 micrometer, 0.45micrometer and 0.22 micrometer in this order to prepare a filter-treatedsolution. Then this filter-treated solution was filtered through anultrafiltration membrane with a fractionating molecular weight of10,000. The filtrate passing through the ultrafiltration membrane wasconcentrated and evaporated to dryness in vacuo and the dried productwas dissolved in water of the amount which was {fraction (1/40)} of thatused for dissolving the pectin in the initial step whereupon aheat-treated pectin solution was prepared.

The heat-treated pectin solution was applied to a column of TOYOPEARLHW-40C (4.4×92 cm; manufactured by Toso) equilibrated with water, a gelfiltration was conducted at a flow rate of 2.5 ml/minute and theapoptosis-inducing activity of each of the fractions was measured by themethod wherein alamarBlue was used as mentioned in Example 8. As aresult, a fraction which was eluted during the eluting time of 448-472minutes showed the activity.

(2) Dα-Glalacturonic acid was dissolved in water to make theconcentration 1% and the solution was adjusted to 7.0 with NaOH. Thiswas heated at 121° C. for 20 minutes and an apoptosis-inducing activityof this heat-treated solution to HL-60 cells was measured by the methodof Example 7 whereupon the heat-treated product showed theapoptosis-inducing activity.

Example 13

Pectin (manufactured by Wako Pure Chemicals; code 167-00542), alginicacid (nonswelling; manufactured by Wako Pure Chemicals; code 011-13341),Dα-galacturonic acid (manufactured by Nacalai Tesque; code 165-18) orD-glucuronic acid (manufactured by Nacalai Tesque; code 169-28) wasdissolved in distilled water to prepare a solution to make theconcentration 1%. In the case of pectin, another solution by dissolvingin an aqueous solution of 1N acetic acid was prepared as well.

Each of those 1% solutions was heated at 121° C. for 30 minutes, 1 hour,2 hours, 4 hours and 16 hours and each of the heated solutions wasadjusted to pH 7 with NaOH and subjected to a sterilization by means ofa filter of 0.22 micrometer to prepare a sample for measuring theapoptosis-inducing activity.

The samples prepared as such were diluted to an extent of 2, 5, 10, 20,50 and 100-fold and their apoptosis-inducing activity was assayed by anMTT method mentioned in Example 11 followed by comparing the resultingactivities. The results are given in Tables 1-5.

(A) The pH of the 1% aqueous solution of pectin was 3.4. The activity ofthe heat-treated pectin was shown in terms of the maximum dilutionswhere the activity was still noted. As shown in Table 1, the activitywas significantly increased by the heating treatment at 120° C. for fourhours.

TABLE 1 Heat Treatment of Aqueous Solution of Pectin Heating pH beforepH after pH after Activity Time Heating Heating Adjustment (Max. Diln.) 2 hrs 3.4 3.3 7.0  2-fold  4 hrs 3.4 3.2 7.2 10-fold 16 hrs 3.4 3.5 7.020-fold

(B) The pH of pectin in a 1% aqueous solution of acetic acid was 2.6.The activity of the solution of pectin in the acetic acid solution wasgiven in terms of the maximum dilution where the activity was stillnoted. As shown in Table 2, the activity was significantly increased byheating at 120° C. for 16 hours.

TABLE 2 Heat Treatment of Pectin-Acetic Acid Solution Heating pH beforepH after pH after Activity Time Heating Heating Adjustment (Max. Diln.) 2 hrs 2.6 2.7 7.0  2-fold  4 hrs 2.6 2.6 7.2  5-fold 16 hrs 2.6 2.8 7.120-fold

(C) The pH of the aqueous solution of galacturonic acid before heatingwas 2.5. The activity of the heat-treated galacturonic acid was shown interms of the maximum dilution where the activity was still noted. Asshown in Table 3, the activity was significantly increased by heating at120° C. for one hour.

TABLE 3 Heat Treatment of Aqueous Solution of Galacturonic Acid HeatingpH before pH after pH after Activity Time Heating Heating Adjustment(Max. Diln.) 30 min 2.5 2.4 6.8  2-fold  1 hr 2.5 2.4 6.9 10-fold  2 hrs2.5 2.4 6.9 20-fold  4 hrs 2.5 2.4 6.8 50-fold 16 hrs 2.5 2.6 6.9100-fold 

(D) The pH of an aqueous solution of glucuronic acid before heating was2.4. The activity of the heat-treated glucuronic acid was given in termsof the maximum dilution where the activity was still noted. As shown inTable 4, the activity significantly increased by heating at 120° C. for30 minutes.

TABLE 4 Heat Treatment of Aqueous Solution of Glucuronic Acid Heating pHbefore pH after pH after Activity Time Heating Heating Adjustment (Max.Diln.) 30 min 2.4 2.6 6.9  10-fold  1 hr 2.4 2.7 6.9  20-fold  2 hrs 2.42.7 6.9  50-fold  4 hrs 2.4 2.6 7.0 100-fold 16 hrs 2.4 2.8 7.0 100-fold

(E) The pH of an aqueous solution of alginic acid before heating was3.3. The activity of the heat-treated alginic acid was given in terms ofthe maximum dilution where the activity was still noted. As shown inTable 5, the activity significantly increased by heating at 120° C. fortwo hours.

TABLE 5 Heat Treatment of Aqueous Solution of Alginic Acid Heating pHbefore pH after pH after Activity Time Heating Heating Adjustment (Max.Diln.)  1 hr 3.3 2.6 6.8  2-fold  2 hrs 3.3 2.5 6.9 10-fold  4 hrs 3.32.7 7.0 10-fold 16 hrs 3.3 2.9 7.3 20-fold

Example 14

Ethanol-washed pectin (manufactured by Wako Pure Chemicals;

code 167-00542) (washed with 80% ethanol, washed with 50% ethanol,washed with 80% ethanol and finally washed with 100% ethanol followed bydrying in vacuo to give a roughly purified pectin in a powdery form),unwashed pectin (manufactured by Wako Pure Chemicals; code 167-00542),alginic acid (nonswelling; D-mannuronic acid type; manufactured by WakoPure Chemicals; code 011-13341), alginic acid (swelling; L-guluronicacid type; manufactured by Wako Pure Chemicals; code 014-13331),D-glucuronic acid (manufactured by Nacalai Tesque; code 169-28) orD-α-galacturonic acid (manufactured by Nacalai Tesque; code 165-18) inan amount of 0.5 g was placed in ten test tubes (one test tube beingunheated used as a control) and heated in air at 120° C., 150° C. or180° C. under a dry condition checking the color change of the sample.Depending upon the color change, samplings were conducted at threepoints and the active ingredient was extracted by the following method.

Thus, each of the dry samples prepared as such was suspended in 12.5 mlof 50% ethanol. The suspension was shaken at room temperature for 16hours and centrifuged to give an extract. The extract was concentratedand dried in vacuo and re-dissolved in distilled water to make theconcentration 1% based upon the amount of the initial sample. The pH ofthe resulting solution was adjusted to around 7 and sterilized by afilter of 0.22 micrometer to prepare a sample for the activitymeasurement. The resulting sample was assayed for the activity by an MTTmethod mentioned in Example 11. The results are given in Tables 6-11together with dry heating temperature, time, pH upon the re-dissolutionand pH after the adjustment. Incidentally, the same operation wasconducted for the unheated sample as well but no activity was noteTables 6-11, the activity is given in terms of the degree of dilution ofthe sample which still exhibited the activity.

From those results, it was found that the active substance was producedby means of a dry heating as well.

TABLE 6 Heat Treatment of EtOH-Washed Pectin Dry Heating Time pH UponRe- pH after Activity Temp (° C.) (min) Dissolution Adjustment (Deg ofDiln) 180  60 3.7 6.9 1 180 120 3.5 6.8 1

TABLE 7 Heat Treatment of Pectin Dry Heating Time pH Upon Re- pH afterActivity Temp (° C.) (min) Dissolution Adjustment (Deg of Diln) 180 1203.9 7.0 1

TABLE 8 Heat Treatment of Alginic Acid (D-Mannuronic Acid Type) DryHeating Time pH Upon Re- pH after Activity Temp (° C.) (min) DissolutionAdjustment (Deg of Diln) 150 40 3.0 6.8 1 150 60 3.0 6.8 1 180 20 3.06.8 1 180 30 3.0 6.8 1 180 40 3.1 6.9 1

TABLE 9 Heat Treatment of Alginic Acid (L-Guluronic Acid Type) DryHeating Time pH Upon Re- pH after Activity Temp (° C.) (min) DissolutionAdjustment (Deg of Diln) 150 60 3.3 6.7 1 180 20 3.3 6.7 1 180 30 3.36.7 1 180 40 3.2 6.8 1

TABLE 10 Heat Treatment of Glucuronic Acid Dry Heating Time pH Upon Re-pH after Activity Temp (° C.) (min) Dissolution Adjustment (Deg of Diln)150 20 3.2 6.8 1 150 30 3.3 6.9 1 150 40 3.3 6.9 1 180 10 3.1 7.0 1 18020 3.3 6.8 1 180 30 3.3 6.9 2

TABLE 11 Heat Treatment of Galacturonic Acid Dry Heating Time pH UponRe- pH after Activity Temp (° C.) (min) Dissolution Adjustment (Deg ofDiln) 120 60 2.9 6.9 1 120 120  2.9 6.9 1 150 20 2.9 6.8 2 150 30 2.96.9 2 150 40 2.9 6.8 2 180 10 2.9 7.1 2 180 20 2.9 6.8 2 180 30 2.9 6.81

Example 15

Commercially available pectin manufactured from apple was dissolved inwater to make the concentration 1% and the solution was placed in apear-shaped flask equipped with a refluxing condenser and heated in anoil bath kept at 110-120° C. for 18 hours, 42 hours or 66 hours.Temperature of the pectin solution during the heating was 100-102° C.

The resulting pectin solution was centrifuged to remove the precipitateand the supernatant was diluted with water to an extent of three- orten-fold to prepare a sample. The diluted sample (10 microliters) and100 microliters of an RPMI 1640 medium containing 10% of fetal bovineserum containing 5,000 HL-60 cells were added to a well of a 96 wellmicrotiter plate and incubated at 37° C. for 48 hours in the presence of5% carbon dioxide gas and the activity was measured by an MTT methodmentioned in Example 11.

The results were that no viable cell was found in the sections to whichthe three-fold diluted solution of pectin heated for 18 hours and alsoto which the three- and ten-fold diluted solutions of pectin heated for42 and 66 hours whereby, at the concentrations of such degrees ofdilution, the pectin heated at 100° C. showed the activity.

On the other hand, in the section to which ten-fold diluted solution ofpectin heated for 18 hours was added, nearly all cells were viable but,when compared with the control which was a section to which water wasadded, the absorbance at 590 nm was lower.

Example 16

Pomosin pectin LM-13CG (manufactured by Hercules) (5 kg) was added to100 liters of tap water, steam was blown thereinto for 35 minutes sothat the liquid temperature was raised from 28° C. to 120° C. and theliquid was kept at 120° C. for five hours with stirring and cooled togive 135 liters of a cooled liquid. To this cooled liquid were added1.35 kg of Celite #545 (manufactured by Celite) and 1.35 kg of Silica#600-S (manufactured by Chuo Silica) as filter aid and the mixture wasfiltered through a compact filter (Advantec #327; with a 6-inch filterpaper in 16 stages) precoated with 0.1 kg of Celite #545 and 0.1 kg ofSilica #600-S. The resulting filtrate was subjected to a continuousinstant heating (at 98° C. for 60 seconds) by a Plate Heater(manufactured by Nichihan Seisakusho) followed by cooling to prepare 150liters of heat-treated pectin solution I.

The pH, acidity and sugar content of the heat-treated pectin solution Iwere about 3.5, 6.2 ml and 5. 8 Brix %, respectively. Incidentally, thepH was measured by a pH-meter, the acidity was given by the amount (ml)of 0.1N NaOH required for neutralizing 10 ml of the sample to pH 7.0 andthe sugar content was measured by a Brix saccharometer.

Activity of the above heat-treated pectin solution I to humanpromyelocytic leukemia cells (HL-60 cells) was measured as follows.

Thus, HL-60 (ATCC CRL-240) was incubated at 37° C. in an RPMI 1640medium (manufactured by Nissui) containing 10% of fetal bovine serum(manufactured by Gibco) treated at 56° C. for 30 minutes and suspendedin the above medium to make the concentration 2.5×10⁵ cells/4.5 ml. To4.5 ml of this suspension was added 0.5 ml of the above heat-treatedpectin solution diluted with water giving the concentration of 20 mg/ml,10 mg/ml, 5 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.2 mg/ml or 0.1 mg/mland the mixture was incubated at 37° C. in the presence of 5% of carbondioxide gas for 24 or 48 hours.

To the cultured cells was added an aqueous solution of trypan blue, themixture was allowed to stand at room temperature for several minutes andan observation was conducted under an optical microscope whereby trypanblue was excreted and colorless cells and blue-colored cells werecounted as viable and dead cells, respectively. The incubated cells werealso observed under an optical microscope whereupon condensation ofnuclei, contraction of cells and production of apoptotic body wereconfirmed in the section to which 1 mg/ml or more heat-treated pectinwas added. Incidentally, in the section to which 0.5 mg/ml or lessheat-treated pectin was added and in the control where 0.5 ml of waterwas added, such phenomena were not noted.

The results are shown in FIG. 12. Thus, FIG. 12 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when the heat-treated pectin solution of various concentrationswas added to the culture medium of HL-60 cells wherein the abscissastands for the incubation time (hours) while the ordinate stands for theviable cell number (×10⁵ cells/5 ml) in the culture medium. In FIG. 12,open square stands for the control where no sample was added, reversedopen triangle stands for the case where 2 mg/ml of heat-treated pectinwas added, closed square stands for the case where 1 mg/ml ofheat-treated pectin was added, closed rhombus stands for the case where0.5 mg/ml of heat-treated pectin was added, closed circle stands for thecase where 0.2 mg/ml of heat-treated pectin was added and closedtriangle stands for the case where 0.1 mg/ml of heat-treated pectin wasadded. Thus, the cases where 50-20 mg/ml of heat-treated pectin wasadded, the similar activity as in the case where 2 mg/ml of heat-treatedpectin was added as shown in a reversed open triangle and, when 1 mg/mlor more of heat-treated pectin was added, an anticancer activity wasnoted.

Example 17

Commercially available D-glucuronic acid (manufactured by Sigma; G5269)was dissolved in water to make the concentration 1% and the solution washeated at 121° C. for four hours, neutralized to pH 7.0 with NaOH anddiluted 10-, 40-, 80- and 160-fold with water. Each of the dilutedheat-treated glucuronic acid solution (0.5 ml) was added to 4.5 ml of anRPMI 1640 medium containing 10% of fetal bovine serum containing 2.5×10⁵HL-60 cells, incubation was conducted at 37° C. for 24 hours in thepresence of 5% carbon dioxide gas and an anticancer activity wasmeasured by the method of Example 7 in terms of an activity forinhibiting the proliferation of the cells. As a result, in the sectionsof 10- to 80-fold diluted solutions were added, a decrease in the cellnumbers and the cell survival rate were noted. In the 40- to 80-folddiluted solutions, DNA was found to become low molecules. Incidentally,survival rate (R) of the cell in terms of % was calculated by thefollowing formula.

R=Vs/(Vs+Ds)×100+Dc/(Vc+Dc)×100

In the formula, Vs and Ds are numbers of vital cells and dead cells,respectively, in the section where the sample was added; and Vc and Dcare numbers of vital and dead cells, respectively, in the section wherewater was added. The anticancer activity in 1 ml of the medium when R is50% is defined as one unit.

When the resulting survival rates of cell were plotted to the commonlogarithmic value of the degree of dilution of the heat-treatedglucuronic acid, all points were on one straight line and the survivalrate R (%) of the heat-treated glucuronic acid was calculated from thefollowing formula.

R=58.656X−31.884

[in the formula, X is a degree of dilution of the heat-treatedglucuronic acid]

From this straight line, it was found that the nondiluted heat-treatedglucuronic acid corresponded to 250 units/ml.

The results are shown in FIG. 13. Thus, FIG. 13 shows the relationshipbetween the degree of dilution and the survival rate of the cells in theculture medium when the heat-treated glucuronic acid with variousdegrees of dilution was added to HL-60 cells followed by incubating for24 hours. The abscissa stands for the degree of dilution (fold; inlogarithm) while the ordinate stands for the survival rate of the cells(%).

Example 18

(1) A 25% solution of puree of peeled rind of apple (manufactured byMaruzen Shokuhin Kogyo), banana puree (manufactured by Ogawa Koryo),green beefsteak plant extract 1/4 (manufactured by Dan Foods), pumpkinextract 60 (manufactured by Dan Foods), minced pumpkin (manufactured byDan Foods), celery puree (manufactured by Dan Foods), burdock puree(manufactured by Dan Foods) or echalote extract 60 (manufactured by DanFoods) was prepared. Each of them was heated at 121° C. for 40 minutes.Each of the solution prepared by the same manner was heated at 121° C.for four hours. Each of the heated solutions was cooled and filtered toprepare a heat-treated solution.

Sugar content and pH of the solution heated at 121° C. for 20 minutesare given in Table 12.

TABLE 12 Sugar Contn Starting Material (Brix) pH Puree of Peeled Rind ofApple 3.6 3.6 Banana Puree 6.0 5.9 Green Beefsteak Plant Extract 1/4 2.25.8 Pumpkin Extract 60 16.8  5.3 Minced Pumpkin 4.0 5.7 Celery Puree 1.65.5 Burdock Puree 2.4 5.8 Echalote Extract 60 15.6  4.9

Sugar content and pH of the solution heated at C121° C. for four hoursare given in Table 13.

TABLE 13 Sugar Contn Starting Material (Brix) pH Puree of Peeled Rind ofApple 3.6 3.6 Banana Puree 5.5 4.6 Green Beefsteak Plant Extract 1/4 2.55.3 Pumpkin Extract 60 16.6  4.7 Minced Pumpkin 3.0 5.0 Celery Puree 1.64.9 Burdock Puree 2.5 4.8 Echalote Extract 60 13.8  4.3

In each of the heat-treated solutions, the fractions having a molecularweight of 10,000 or less were found to show an anticancer activity asmentioned in Example 17.

Then sugar content (Brix) was adjusted to 1 and an organoleptic test wasconducted for each of the heat-treated solutions whereby all of theheat-treated solutions showed good organoleptic property as food orbeverage.

(2) The 25% aqueous solutions of banana puree, apple puree and celerypuree heated at 121 ° C. for four hours were taken as representativeexamples and their anticancer activity units were measured by the methodof Example 17. The results are given in Table 14. Thus, as a result ofthe heating treatment, anticancer active substance was produced in eachof the treated solutions.

TABLE 14 Puree Used Activity (units/ml) Banana Puree 23.4  Apple Puree9.5 Celery Puree 0.5

Water (160 ml) was added to 40 g of (1) radish leaves, (2) carrotleaves, (3) carrot, (4) cabbage, (5) eggplant without rind, (6) bananaor (7) albedo of hassaku orange and each of the mixtures was homogenizedusing a mixer. A part of it was heated at 121° C. for four hours andcentrifuged and the supernatant thereof was adjusted to pH 6 with NaOHto prepare a sample A while the remainder was adjusted to pH 3 with HCland heated at 121° C. for four hours and the supernatant after thecentrifugation was adjusted to pH 6 with NaOH to prepare a sample B.

Each of the samples A and B prepared from (1)-(7) was diluted and 10microliters of the diluted solution was subjected to a measurement foranticancer activity by an MTT method mentioned in Example 11. Theresults are given in Table 15. The data shown in Table 15 are thedegrees of dilution where the activity was still noted and the sign “−”shows that no activity was noted in the sections to which nondilutedsolution was added. In all of the fruits and the vegetables, generationof the activity was noted in their heat-treated products. In the table,the degrees of dilution are those where the cells were completely killedwhile the values in parentheses are those where the cells were affected.

TABLE 15 Vegetables and Degree of Dilution for Fruits Used Sample ASample B Radish Leaves 1 (4) 2 (4) Carrot Leaves — 1 Carrot 2 (4) 1 (2)Cabbage 1 (4) 1 Eggplant 1 (2) 1 Banana 2 (4) 2 (4) Wave Packet 4 (8) 4(8)

Example 20

Nonswelling alginic acid (manufactured by Wako Pure Chemicals;011-13341) or swelling alginic acid (manufactured by Wako PureChemicals; 014-13331) was suspended in water to make the concentration1% whereupon the pH was 3.32 and 3.38, respectively. Each of them washeated at 121° C. for 20 minutes and its anticancer activity wasmeasured as a cell proliferation inhibiting activity to HL-60 cells bythe method of Example 7.

Incidentally, the HL-60 cell numbers at the initiation of the incubationwere 3×10⁵ cells/5 ml.

The results are shown in FIG. 14. Thus, FIG. 14 shows the relationshipbetween the incubation time and the viable cell number in the culturemedium when the heat-treated nonswelling alginic acid or swellingalginic acid solution was added to the culture medium of HL-60 cells tomake the concentration 1 mg/ml. The abscissa stands for the incubationtime (hours) while the ordinate stands for the viable cell number (×10⁵cells/5 ml) in the culture medium. In FIG. 14, open square stands forthe control where no sample was added, open rhombus stands for the casewhere the heat-treated nonswelling alginic acid was added and opentriangle stands for the case where the heat-treated swelling alginicacid was added. Thus, a high activity was noted in the heat-treatednonswelling alginic acid.

Example 21

A 1% aqueous suspension of alginic acid HFD (manufactured by DainipponPharmaceutical) was prepared and subjected to a heat treatment at 120°C. for four hours. The supernatant of the heat-treated solution aftercentrifugation was subjected to an anticancer activity measurement bythe method mentioned in Example 17 to calculate the anticancer activityunit. The results are shown in Table 16. Thus, generation of an activesubstance was noted in the heat-treated alginic acid.

TABLE 16 Heat-Treated Alginic Acid HFD Activity (Units/ml) Heated 1%Solution 83.3

Example 22

Alginic acid HFD (manufactured by Dainippon Pharmaceutical) (1 g) wassuspended in 50 ml of water and heated at 121 ° C. for 30 minutes, 1hour, 2 hours or 14 hours. Each of the heat-treated solutions wasprepared by means of centrifugation and its molecular weight wasdetermined. Determination of the molecular weight was conducted underthe following conditions.

Guard Column: TSK Guard Column PWH

Column: TSK Gel G3000PW

Eluting Solution: 0.2M NaCl

Detection: by absorption at 210 nm

When the heating time was 30 minutes, 1 hour, 2 hours, 4 hours and 14hours, the low-molecular weight decomposed products of the molecularweights of 1,800; 1,200 and 630; 1,100 and 630; 1,100 and 630; and 620and 400 as the main peaks were produced respectively and, at the sametime, other low-molecular weight decomposed products were produced aswell. Incidentally, no high-molecular weight substances having themolecular weight of 10,000 or more were contained and the anticancer andantibacterial activities were found in the fractions having a molecularweight of 500 or less.

Example 23

(1) Commercially available glucuronolactone (manufactured by Merck; CodeNo. 100282) was dissolved in water to make the concentration 1% and thesolution was heated at 121° C. for 0.5, 1, 2, 4 or 16 hours. Theanticancer activity of each of the heated solutions prepared as such wasmeasured by the method of Example 17. In the solution heated for 0.5hour, production of the anticancer substance was noted. It was foundthat the longer the heating time, the more the production of theanticancer substance and, in each of the products heated for 4 and 16hours, the production was about 10-fold of that heated for 0.5 hour.

(2) The above-mentioned glucuronolactone was dissolved in water to makethe concentration 0.1%, 1%, 2%, 5%, 10% or 20% and each of the solutionwas heated at 121° C. for four hours. The anticancer activity of each ofthe heat treated solutions was measured by the method of Example 17.Although the production of anticancer substance was noted in all of theconcentrations, the potency of the anticancer activity of theheat-treated product per the glucuronolactone used was the highest when0.1% aqueous solution of glucuronolactone was used.

(3) The pH of the above-mentioned 1% aqueous solution ofglucuronolactone was adjusted to 1, 2, 3 or 4.5 with HCl or with NaOHand each of the solutions was heated at 121° C. for four hours. Theanticancer activity of each of the heat-treated solutions prepared assuch was measured by the method of Example 17. Although the productionof anticancer substance was noted in all cases of the above pH values,the potency of the anticancer activity of the heat-treated product at pH3-4.5 was about 15-fold of that at pH 1 per the glucuronolactone used.

(4) Commercially available D-glucuronic acid (manufactured by Sigma;G5269) was dissolved in water to make the concentration 1% and heated at121° C. for four hours whereby a sample (pH: 2.6) where the pH was notadjusted and another sample where the pH was adjusted to 6.6 with NaOH.Each 1 ml of them was stored at −20° C., 4° C. and 37° C. and theanticancer activity was measured by the method of Example 17.

The result after storing for 25 days was that, when stored at 37° C.,the anticancer activity of the heat-treated product was somewhatdecreased while, in the case of 4° C. and −20° C., the activity wasalmost stable.

Example 24

Pomosin pectin type LM-13CG (manufactured by Hercules), alginic acid HFD(manufactured by Dainippon Pharmaceutical), D-glucuronic acid(manufactured by Nacalai Tesque) or glucuronolactone (manufactured byMerck) was dissolved or suspended in water to make the concentration 1%and the solution or the suspension was heated at 95° C., 121° C. or 132°C. for 16 hours. The anticancer activity units of those heat-treatedproducts were measured by the method of Example 17. The results aregiven in Table 17.

TABLE 17 Heated Material Heating Temp (° C.) Activity (Units/ml) Pectin95 1.2 121 32.3 132 1.4 Alginic Acid 95 1.0 121 57.8 132 25.7 GlucuronicAcid 95 40.8 121 345 132 30.2 Glucuronolactone 95 42.7 121 5,376 13233.8

Example 25

(1) Apple pectin (1.5 g; manufactured by Wako Pure Chemicals) wassuspended in 100 ml of water and the suspension was adjusted to pH 12with NaOH. This was stirred at 4° C. keeping the pH at 12 by a gradualaddition of NaOH. When eight hours elapsed after that, a decrease in pHwas not observed. After 24 hours, the suspension was adjusted to pH 5with HCl, 4-fold by volume of ethanol was added thereto and the mixturewas stirred at 4° C. for one hour and filtered through a filter paper.The resulting precipitate was washed with 65% ethanol and then with 99.5% ethanol followed by drying in vacuo to give 1.32 g of pectic acid.

(2) Pectic acid (200 mg) obtained in the above (1) was dissolved in 200ml of water and 2 ml of concentrated HCl was gradually added thereto.The mixture was heated at 80° C. for 66 hours and centrifuged at20,000×g for 30 minutes to give a supernatant and a precipitate. Thesupernatant was adjusted to pH 7 by NaOH, dialyzed against water using adialyzing membrane with cutoff molecular weight of 1000 and dried byfreezing to give 18.4 mg of an acid-soluble fraction. The precipitatewas suspended in 30 ml of water, adjusted to pH 6 by NaOH, dialyzedagainst water using a dialyzing membrane with cutoff molecular weight of1000 and freeze-dried to give-114 mg of an acid-insoluble fraction.

(3) Each of the acid-soluble and acid-insoluble fractions obtained inthe above (2) was dissolved in water to prepare a 1% solution and thesolution was adjusted to pH 3 with HCl and heated at 121° C. for 20minutes. Anticancer activity of the resulting heat-treated products wasdetermined by measuring an activity for inhibiting the cellproliferation by means of a method using alamarBlue as mentioned inExample 2. As a result, an anticancer activity was noted in theacid-soluble fraction of the heat-treated product.

Example 26

(A) D-Glucuronic acid (manufactured by Nacalai Tesque, code 169-28) wasdissolved in distilled water to make the concentration 1%, the solutionwas heated at 120° C. overnight and the pH was adjusted to around 7 byNaOH. Antibacterial activity of this heat-treated glucuronic acid wasinvestigated as follows.

Thus, the microorganism to be tested was subjected to a seed culture inan L-broth (containing 1% of tryptone, 0.5% of yeast extract and 0.5% ofNaCl; pH: 7.0) overnight. A seed-cultured liquid (5 microliters) wasinoculated to a medium prepared by adding none of or 50, 100, 250, 500or 1000 microliters of heat-treated glucuronic acid to 5 ml of L-brothand the culture was incubated at 37° C. with shaking whereupon thegrowth was observed. At the initiation of the incubation and at eighthours thereafter, turbidity of the culture was measured using a FujiDigital Turbidimeter (sold by Fuji Kogyo KK; manufactured by AkiyamaDenki Seisakusho) under the condition that the adjusting scale was 82.3and, by means of the value (growth turbidity) obtained by subtractingthe value at the initiation stage from the value after eight hours,growth of the test microorganism was determined. Incidentally, in thecase of the test organism (6), a brain heart infusion medium was usedinstead of the L-broth.

The microorganisms tested were Escherichia coli HB 101 (ATCC 33694; testmicroorganism (1)); Salmonella typhimurium LT-2 (ATCC 27106; testmicroorganism (2)); Pseudomonas aeruginosa (IFO 3080; test microorganism(3)); Staphylococcus aureus 3A (NCTC 8319; test microorganism (4));Bacillus subtilis (IFO 3034; test microorganism (5)); and Streptococcusmutans GS5 (a strain stored at the National Institute of Health; testmicroorganism (6)).

TABLE 18 (Growth Turbidity) Amount of Heat-Treated Product Test (μl/5 mlmedium) Microorganism 0 50 100 250 500 (1) 239 183  89  6 10 (2) 247 177 36  5 11 (3) 273 262 212 237 61 (4) 285 251 247  20 11 (5) 280 258 205 73 13 (6) 140 136 131 125 10

The heat-treated product showed antibacterial activity to each of thetest microorganisms at any of the additions of 100-500 microliters/5 ml.In addition, the heat-treated product showed antibacterial activity tomethicillin-resistant Staphylococcus aureus, enterotoxin-productive S.aureus, Bacillus cereus of a vomiting type, B. cereus of a diahhrea typeand enterorrhagiac E. coli O-157 as well.

(B) Alginic acid for food additive (Alginic Acid HFD; manufactured byDainippon Pharmaceutical Co., Ltd. ) was dissolved in distilled water tomake the concentration 1% and the solution was heated at 120° C.overnight and adjusted to pH around 7 with NaOH. Like in theabove-mentioned method, this heat-treated alginic acid solution wasadded in an amount of 250 to 1000 microliters and its antibacterialactivity to the test microorganisms (1)-(6) was tested. In the case ofthe test microorganism (6), the solution was added to an extent of 1500microliters. The results are given in Table 19.

TABLE 19 (Growth Turbidity) Amount of Heat-Treated Product Test (μl/5 mlmedium) Microorganism 0 250 500 1000 1500 (1) 239  30  8 13 — (2) 247 10  8 12 — (3) 273 233 188 30 — (4) 285 222  12 15 — (5) 280 158  22 13— (6) 140 138 130 101  12

The heat-treated product showed antibacterial activity to each of thetest microorganisms at any of the additions of 250-1500 microliters/5ml. In addition, the heat-treated product showed antibacterial activityto methicillin-resistant Staphylococcus aureus, enterotoxin-productiveS. aureus, Bacillus cereus of a vomiting type, B. cereus of a diahhreatype and enterorrhagiac E. coli O-157 as well.

Example 27

Commercially available apple pectin (5 g) was dissolved in 500 ml of200mM NaCl and adjusted to pH 7.0 with NaOH. This solution was heated at121° C. for 30 minutes and readjusted to pH 7.0 with NaOH. This wascentrifuged at 12,000 rpm (about 10,000 g) for 30 minutes and theanticancer action of the re supernatant (hereinafter, referred to as“the sample”) was tested.

Murine solid carcinoma Meth A (4×10⁶ cells/mouse) was subcutaneouslyinjected to the abdominal region of a BALB/c mouse of ten weeks age(female; body weight ca. 20 grams). After that, the sample (100mg/kg/day) was subcutaneously injected into the same place forconsecutive ten days.

On the other hand, a physiological saline solution instead of the samplewas subcutaneously injected to the control group in the same manner.After two weeks, the solid carcinoma tissue formed in the abdominalregion of the mouse was excised and its weight was measured. The resultsare given in Table 20. Thus, in the control group, an average weight ofthe carcinoma was 1.26 g while, in the group administered with thesample, it was 0.88 g whereby the inhibition rate to cancer was about30.1% and an anticancer action was noted in the sample.

TABLE 20 Weight of Excised Inhibiting Carcinoma (grams) Rate (%) ControlGroup 1.23 1.21 1.34 1.52 1.74 1.15 1.09 0.76 1.26 ± 0.10 in average 0% Group Administered with the Sample 1.69 1.61 0.33 0.14 0.17 0.99 1.210.88 ± 0.25 in average 30.1%

Example 28

Murine leukemia cell line P-388 (1×10⁶ cells/ml) was incubated in vitrofor six hours together with the sample (1 mg/ml) prepared in Example 27in an RPMI 1640 medium containing 10% fetal bovine serum and, afterthat, 1 ml/mouse of the resulting one was intraperitoneally injected asit was to a DBA/2 mouse of five weeks age (female; body weight ca. 20grams). (P-388:1×10⁶cells/mouse; the sample: 50 mg/kg)

On the other hand, in the control group, P-388 incubated under the samecondition was injected into the mouse together with the physiologicalsaline solution instead of the sample.

In the two groups (each group comprising eight mice), survived numbers,average survived days and survival rate were calculated and the resultsare given in FIG. 15. Thus, FIG. 15 shows an anticancer action of thesample to leukemia cells in which abscissa and ordinate are surviveddays and survived numbers, respectively, of the mice. In the figure, abroken line and a solid line are the control group and the groupadministered with the sample, respectively. Thus, in the control group,average survived days are 8.0 days while, in the group administered withthe sample, average survived days are 14.6 days whereby the survivalrate is 182.5% and a significant surviving effect was noted in thesample.

In the experiments which were conducted at the same time, there was nodifference in terms of the survival rate of P-388 cells after an invitro incubation for six hours between the group to which the sample wasadded and not added and the cell survival rate was 100% in both groups.

Example 29

Galacturonic acid or glucuronic acid was dissolved in distilled water tomake the concentration 50 mg/ml and the solution was heated at 121° C.for 20 minutes and adjusted to pH7.0 with1NaOH. This was diluted with aphysiological saline solution to a desired concentration and subjectedto the following tests.

(1) Meth A cells (4×10⁶ cells/mouse) were subcutaneously injected to theabdominal region of a BALB/c mouse of eight weeks age (female; bodyweight ca. 20 grams). After that, the heat-treated galacturonic acid(100 mg/kg/day) or heat-treated glucuronic acid (100 mg/kg/day) wassubcutaneously injected to the same place for consecutive ten days.

After two weeks, the carcinoma tissue formed in the abdominal region ofthe mouse was excised and its weight was measured. The results are givenin Table 21. Thus, in the control group, the average weight of carcinomawas 1.48 g while, in the groups administered with the heat-treatedgalacturonic acid and with the heat-treated glucuronic acid, the averageweights were 0.94 g and 0.86 g, respectively whereby the inhibitionrates were 26.5% and 41.9%, respectively. Thus, significant anticanceraction (p<0.05 to the control group) was noted in both groups.

TABLE 21 Numbers Carcinoma Wt (g) Inhibition of Mice (average ± SD) RateControl Group 8 1.48 ± 0.54 — Group Administered with Heat-TreatedGalacturonic 6 0.94 ± 0.25 26.5% Acid Heat-Treated Glucuronic acid 70.86 ± 0.31 41.9%

(2) Sarcoma-180 (5.5×10⁶ cells/mouse) was subcutaneously injected intothe abdominal region of 16 female ICR mice (body weight: ca. 26 grams)of six weeks age and divided into eight mice for control group and eightmice for group administered with heat-treated glucuronic acid.

The group administered with heat-treated glucuronic acid was freely fedwith the heat-treated glucuronic acid from a water-supplying bottlewhere the acid was diluted with tap water so as to make the dose of theheat-treated glucuronic acid about 1 g/kg/day. In the control group, tapwater was given by the same manner. With respect to a feed, both groupswere allowed to take it freely during the term of the experiment.

The survived numbers after 35 days from the subcutaneous injection ofSarcoma-180 were two out of eight in the control group while they wereeight out of eight in the group administered with the heat-treatedglucuronic acid. Thus, a remarkable survival effect by oraladministration of heat-treated glucuronic acid was noted.

Example 30

Murine leukemia cell line P-388 (1×10⁶ cells/ml) was incubated for sixhours in vitro in an RPMI 1640 medium containing 10% fetal bovine serumtogether with a heat-treated galacturonic acid (1 mg/ml) or aheat-treated glucuronic acid (1 mg/ml) prepared in Example 29 and then 1ml of it was intraperitoneally injected to DBA/2 mouse (female; bodyweight ca. 20 grams) (P-388: 1×10⁶ cells/mouse; heat-treated acid: 50mg/kg). To the control group were injected P-388 cells (1×10⁶cells/mouse) incubated under the same condition together with aphysiological saline solution. Incidentally, in the experiment conductedat the same time, there was no difference in terms of the survival rateof the P-388 cells after an in vitro incubation for six hours betweenthe group administered with a heat-treated acid and that with aphysiological saline solution and the survival rates were 100% in bothgroups.

Each eight mice were used for each group and the average survived daysand survival rate were calculated from the survived numbers of the mice.

The result are shown in FIG. 16. Thus, FIG. 16 shows the relationshipbetween the days after transplantation of the P-388 cells and thesurvived numbers of mice in each of the groups where an ordinate showssurvived numbers of mice while an abscissa shows the survived days ofmice. In the figure, a solid line, a broken line and a two-dot chainline show the control group, the group administered with theheat-treated galacturonic acid and the group administered with theheat-treated glucuronic acid, respectively.

As calculated from the results of FIG. 16, the average survived days was11.4 days in the control group while, in the group administered with theheat-treated galacturonic acid (50 mg/kg), the average survived dayswere 23.5 days or more and the survival rate was 206.1% or more on 24thdays after the transplantation of the cells and, in the groupadministered with the heat-treated glucuronic acid (50 mg/kg), theaverage survived days were 16.8 days and the survival rate as 147.3%whereby significant surviving effect was noted as compared with thecontrol group.

Example 31

D-Glucuronic acid (10 g) (G5269 manufactured by Sigma) was dissolved inone liter of water, heated at 121° C. for four hours and neutralized topH 7.0 with NaOH.

The heat-treated product (500, 5 or 0.05 micrograms/ml) was added to anRPMI 1640 medium containing 10% of fetal bovine serum containing1×10⁵/ml of HL-60 cells (ATCC CCL-240) and was incubated at 37° C. forthree days in the presence of 5% carbon dioxide gas. Then a part of theincubated cells were smeared on a slide glass, subjected to aWright-Giemsa stain mentioned in page 191 of “Tissue Culture Techniques”(edited by Japan Tissue Culture Society, published by Asakura Shoten,1982) and the degree of differentiation was observed under an opticalmicroscope. The result was that depending upon the concentration of theheat-treated glucuronic acid which was added thereto, the cancer cellswere differentiated to monocytes or to macrophage-like cells and theratio of the mature bone marrow cells in the incubated cells wasincreased. The results are shown in FIG. 17. Thus, FIG. 17 shows therelationship between the incubating time and ratio of the mature bonemarrow cells in the incubated cells where the abscissa and the ordinateshows the incubated time (days) and the ratio (%) of the mature bonemarrow in the incubated cells, respectively. In FIG. 17, open squareshows the group where no sample was added (control); open rhombus showsthe group where 500 micrograms/ml of heat-treated glucuronic acid wasadded; open circle shows the group where 5 micrograms/ml of heat-treatedglucuronic acid was added; and open triangle shows the group where 0.05microgram/ml of heat-treated glucuronic acid was added.

Example 32

Antiulcer Action of Heat-Treated Glucuronic Acid.

D-Glucuronic acid (G5269 manufactured by Sigma) was dissolved indistilled water to make the concentration 10 mg/ml, heated at 121° C.for four hours, adjusted to pH 7.0 with1NaOH and concentrated to 200mg/ml by means of a freeze-drying to prepare a heat-treated glucuronicacid concentrate. This was subjected to the following experiments.

Wistar strain rats (body weight: 220-275 grams) were fasted for 24 hoursand, since three hours before the initiation of the experiment, no waterwas given to them.

One ml of 99.5% ethanol was orally given to a rat and, one hourthereafter, stomach was excised under anesthetization with ether.Pylorus and cardia of the excised stomach were ligated, a 1% formalinsolution was infused and the stomach was immersed in said solution forten minutes. Then the stomach was cut out along a greater curvature andthe length (mm) of the tumor generated in the stomach gland region wasmeasured.

In the group administered with heat-treated glucuronic acid, theabove-mentioned heat-treated glucuronic acid concentrate was orallygiven at the rate of 1 g/kg before 30 minutes of administration ofethanol. Distilled water was given to the control group by the samemanner.

Length of the ulcer after one hour from the administration of ethanolwas 78.2±28.5 mm (average±standard deviation) in the control group (N=6)while, in the group (N=3) administered with the heat-treated glucuronicacid, no ulcer was noted at all whereby a remarkable antiulcerativeaction was noted.

Example 33 Injection

The sample prepared by evaporation of the ethanol-treated supernatantfraction as mentioned in Example 8 was dissolved in distilled water forinjection to prepare a 1% solution. This solution was packed in vialsfor freeze-drying in an amount of 10 mg/vial based upon theabove-mentioned sample from the supernatant fraction and thenfreeze-dried. A physiological saline solution (2 ml) was separatelyattached thereto as a solvent for dissolution.

Example 34 Injection

Galacturonic acid was dissolved in distilled water for injection to makethe concentration 10 mg/ml, heated at 121° C. for 20 minutes, cooled andneutralized to prepare a neutral solution of the heat-treated acid. Thissolution was packed in vials for freeze-drying in an amount of 50 mgbased upon the dried heat-treated acid and then freeze-dried. Aphysiological saline solution (2 ml) was separately attached thereto asa solvent for dissolution.

Example 35 Tablets

Tablets were prepared in accordance with the following formulation.

Heat-treated pectic acid 10 mg Corn starch 65 mg Carboxymethylcellulose20 mg Polyvinylpyrrolidone 3 mg Magnesium stearate 2 mg Total 100 mg pertablet

Pectin was heated by the method mentioned in Example 7, neutralized,freeze-dried and the resulting freeze-dried product was used as theheat-treated pectin.

Example 36

Green tea was prepared according to a conventional method using 10 g ofgreen tea leaves, 0.2 g of vitamin C and 1,000 ml of deionized water.The heat-treated pectin solution I mentioned in Example 16 was added inan amount of 50 mg (based upon a solid) to 100 ml of the productwhereupon the product (1) of the present invention was prepared. Thecontrol was that to which nothing was added. An organoleptic evaluation(by a five-point method where point 5 was good and point 1 was bad) wasconducted by 20 panelists and the averages of the results are shown inTable 22.

TABLE 22. Organoleptic Evaluation Product (1) Control Breadth of Taste4.1 3.2 Balance of Taste 3.8 3.4 Total Taste 4.1 3.3

From Table 22, the evaluation was that, as compared with the control,the product (1) of the present invention had wider and broader taste andwell-balanced taste whereupon flavor and taste of the tea were improvedand an effect of “a hidden flavor” was achieved.

Example 37

An alcoholic beverage was prepared by a conventional method inaccordance with a compounding as shown in Table 23.

TABLE 23 Table of Compounding Frozen concentrated juice of Citrus unshiu(45 Brix degree) 110 g Granulated sugar 80 g Citric acid 2 g Sodiumcitrate 0.5 g Orange essence 2 g 5% (v/v) Aqueous solution of alcoholbalance Total 1,000 ml Note: The beverage prepared as such was cooled at5° C. and then carbonic acid was made contained therein by means of asoda siphon.

The heat-treated pectin solution I mentioned in Example 16 was added inan amount of 45 mg (based upon a solid) to 100 ml of the productwhereupon the product (2) of the present invention was prepared. Thecontrol was that to which nothing was added. An organoleptic evaluationwas conducted by the same manner as in Example 36. The results are givenin Table 24.

TABLE 24 Organoleptic Evaluation Product (2) Control Breadth of Taste3.9 3.3 Balance of Taste 4.0 2.7 Total Taste 3.9 3.0

As shown in Table 24, it was noted that, as compared with the control,the product (2) of the present invention had wider and broader taste.Particularly in this product (2), the acidic taste became milder and thefinish was that the flavor and taste of the large mandarin (Citrusunshiu) were enhanced.

Example 38

The product (3) of the present invention was prepared from aconventionally-prepared sake (Japanese rice wine) by adding theheat-treated pectin solution II of Example 9 in an amount of 35 mg (as asolid) per 100 ml of the final product. A product to which noheat-treated pectin solution was added was used as a control.

The organoleptic evaluation was conducted by the same manner as inExample 36. Aroma and feel on the tongue were added to the evaluatingitems and the results are given in Table 25.

TABLE 25 Organoleptic Evaluation Product (3) Control Breadth of Taste3.8 3.0 Balance of Taste 3.4 2.9 Aroma 2.9 2.9 Feel on the TongueMildness 3.8 2.6 Smoothness 4.0 2.9 Total Taste 3.6 2.8

As shown in Table 25, it was noted that, as compared with the control,the product (3) of the present invention had wider and broader taste andimproved feel on the tongue and accordingly that the taste and the feelupon drinking as table luxuries were improved.

Example 39

The product (4) (mirin—a sweet sake) and the product (5) (fermentedseasoning) of the present invention were prepared from theconventionally-prepared mirin and fermented seasoning by adding theheat-treated pectin solution I of Example 16 in an amount of 40 mg (as asolid) per 100 ml of each of the final products. Products to which noheat-treated pectin solution were added was used as controls.

The organoleptic evaluation was conducted by the same manner as inExample 36. The results are given in Table 26.

TABLE 26 Organoleptic Evaluation Mirin Fermented Seasoning Product (4)Control Product (5) Control Breadth of Taste 3.8 3.0 2.9 2.4 Balance ofTaste 3.5 3.0 2.7 2.1 Total Taste 3.6 3.1 2.8 2.2

As shown in Table 26, it was noted that, as compared with each of thecontrols, the products (4) and (5) of the present invention showedimprovements in the balance and the breadth of the taste and accordinglythat seasonings having a deep taste can be prepared.

Example 40

Fish powder (4.7 kg), 0.8 kg of sea algae, 2.5 kg of sesame, 1.0 kg ofsalt and 0.5 kg of sodium glutamate were mixed and the mixture wasgranulated by a conventional method to prepare furikake (seasoned fishflour).

A product (6) of the present invention was prepared by adding 1,000 mg(as a solid) of the heat-treated pectin solution II of Example 9 per 100g of the product. No heat-treated pectin solution was added was used asa control. Those were sprinkled on boiled rice and the organolepticevaluation in terms of feel on eating was conducted by the same manneras in Example 36.

The result was that, as compared with the control, the product (6) ofthe present invention well fitted the boiled rice in the mouth, had awell-balanced taste and a mild finish and, as a whole, exhibited animproved quality as a furikake.

Example 41

A beverage was prepared using the heat-treated vegetable and fruits. Thecompounding is shown in Table 27.

TABLE 27 Carrot (rhizome) 200 g Pineapple (fruit) 500 g Banana (fruit)500 g Granulated sugar 76 g Anhydrous citric acid 2 g Water balanceTotal 2000 g

Each of carrot, pineapple and banana in the compounding as shown inTable 27 was well stirred and disintegrated using a commerciallyavailable mixer to prepare puree of each of them. Then each of thosepurees was heated at 121° C. for four hours in a tightly closed stateand, after that, they were mixed in accordance with the above table toprepare a beverage of the present invention.

On the other hand, each of those vegetable/fruits were not heated buttheir disintegrated product was just mixed according to the above tableto prepare a control beverage. Organoleptic evaluation of the product ofthe present invention and the control was conducted by the same manneras in Example 36. The results are shown in Table 28.

TABLE 28 Organoleptic Evaluation (Average Values) Product of theInvention Control Aroma 3.5 3.0 Taste 4.0 2.6 Texture 4.3 3.2 TotalEvaluation 4.0 2.8 Comments Mild; well-mixed No mild feel; separatedtaste; united feel tastes; aroma was not of aroma; mild well-balanced;and a bit feel on the tongue rough on the tongue

From Table 28, it was noted that, as compared with the control, theproduct of the present invention had a mild feel, showed a well-mixedtaste, had a united feel of aroma and exhibited a mild feel on thetongue whereby an appreciable beverage was prepared.

The pharmaceutical agent of the present invention can be used as atherapeutic agent for infectious diseases, lowered or risen immunefunction, cancerous diseases, viral diseases, ulcer, peridontaldiseases, and the like. Further, an apoptosis-inducing method of thepresent invention is useful in studying the relation between apoptosisand defensive mechanism of living body, immune function and cancerousand viral diseases and also in developing the inhibitors for inductionof apoptosis. Particularly, the saccharide compounds of the presentinvention in edible products have a long history as food and theheat-treated product of the present invention prepared from them is of avery high safety when given orally. In addition, it is a matter ofcourse that the food or beverage containing the heat-treated product ofthe present invention and the food, beverage or antiseptic agent forfood or beverage prepared by adding and/or diluting the heat-treatedproduct of the present invention are of high safety and, due to theirapoptosis-inducing action, anticancer action, antiangiogenic action,antiviral action, antiulcer action, and the like, they are very usefulfor prevention and therapy of gastrointestinal cancer, viral diseasesuch as cold by influenza virus, ulcer, and the like, and also forimprovement of hepatic function.

As mentioned hereinabove, the heat-treated product of the presentinvention can be easily manufactured in a low cost and, when it is usedas an additive to food or beverage, it can give various physiologicalfunctions, antibacterial action, apoptosis-inducing action, anticanceraction, antiviral action, and the like, due to its various physiologicalfunctions whereby the heat-treated product of the present invention isquite useful as an additive to food or beverage, particularly as anantiseptic agent for food and beverage.

What is claimed is:
 1. A method of inducing apoptosis comprisingadministering, as an effective component, a product obtained by heatinga substance selected from the group consisting of (a) an uronic acid oruronic acid derivative, (b) a saccharide compound comprising an uronicacid or uronic acid derivative, (c) a substance comprising a saccharidecompound comprising an uronic acid or uronic acid derivative and (d) amixture thereof.
 2. The method according to claim 1, wherein the uronicacid is galacturonic acid, glucuronic acid, guluronic acid, mannuronicacid or iduronic acid.
 3. The method according to claim 1, wherein theuronic acid derivative is selected from the group consisting of uronicacid lactone, uronic acid ester, uronic acid amide and salts thereof. 4.The method according to claim 1, wherein the saccharide compound isselected from the group consisting of pectin, pectic acid, alginic acid,hyaluronic acid, heparin, fucoidan chondroitin sulfate, chondroitin,dermatan sulfate and decomposed products thereof.
 5. The methodaccording to any one of claims 1 and 2-4, wherein the substance isheated at 60-350° C. for several seconds to several days.
 6. The methodaccording to any one of claims 1 and 2-4, wherein the substance isheated under acidic to neutral conditions.
 7. The method according toclaim 1, wherein the product is further obtained by molecular weightfractionation of the substance after heating.
 8. A method of inducingapoptosis comprising administering, as an effective component, a productobtained by heating a substance selected from the group consisting of(a) an uronic acid or uronic acid derivative, (b) a saccharide compoundcomprising an uronic acid or uronic acid derivative, (c) a substancecomprising a saccharide compound comprising an uronic acid or uronicacid derivative and (d) a mixture thereof, and molecular weightfractionating the heated substance to obtain the product.